Structure-based designed herbicide resistant products

ABSTRACT

Disclosed herein are structure-based modelling methods for the preparation of acetohydroxy acid synthase (AHAS) variants, including those that exhibit selectively increased resistance to herbicides such as imidazoline herbicides and AHAS inhibiting herbicides. The invention encompasses isolated DNAs encoding such variants, vectors that include the DNAs, and methods for producing the variant polypeptides and herbicide resistant plants containing specific AHAS gene mutations. Methods for weed control in crops are also provided.

This application is a 371 of PCT/US96/05782 filed Apr. 19, 1996, whichis a continuation-in-part of U.S. Ser. No. 08/455,355 filed on May 31,1995 issued as U.S. Pat. No. 5,928,937, which is a Division of Ser. No.08/426,125 filed Apr. 20, 1995, issued as U.S. Pat. No. 5,853,973.

FIELD OF THE INVENTION

This invention pertains to structure-based modelling and design ofvariants of acetohydroxy acid synthase (AHAS) that are resistant toimidazolinones and other herbicides, the AHAS inhibiting herbicides,AHAS variants themselves, DNA encoding these variants, plants expressingthese variants, and methods of weed management.

BACKGROUND OF THE INVENTION

Acetohydroxy acid synthase (AHAS) is an enzyme that catalyzes theinitial step in the biosynthesis of isoleucine, leucine, and valine inbacteria, yeast, and plants. For example, the mature AHAS from Zea maysis approximately a 599-amino acid protein that is localized in thechloroplast (see FIG. 1; SEQ ID NO:1). The enzyme utilizes thiaminepyrophosphate (TPP) and flavin adenine dinucleotide (FAD) as cofactorsand pyruvate as a substrate to form acetolactate. The enzyme alsocatalyzes the condensation of pyruvate and 2-ketobutyrate to formacetohydroxybutyrate. AHAS is also known as acetolactate synthase oracetolactate pyruvate lyase (carboxylating), and is designated EC4.1.3.18. The active enzyme is probably at least a homodimer. Ibdah etal. (Protein Science, 3:479-S, 1994), in an abstract, disclose one modelfor the active site of AHAS.

A variety of herbicides including imidazolinone compounds such asimazethapyr (PURSUIT®—American Cyanamid Company-Wayne, N.J.),sulfonylurea-based compounds such as sulfometuron methyl (OUST®—E.I. duPont de Nemours and Company-Wilmington, Del.), triazolopyrimidinesulfonamides (Broadstrike™—Dow Elanco; see Gerwick, et al., Pestic. Sci.29:357-364, 1990), sulfamoylureas (Rodaway et al., Mechanisms ofSelectively of Ac 322,140 in Paddy Rice, Wheat and Barley, Proceedingsof the Brighton Crop Protection Conference-Weeds, 1993),pyrimidyl-oxy-benzoic acids (STABLE®—Kumiai Chemical Industry Company,E.I. du Pont de Nemours and Company; see, The Pesticide Manual 10th Ed.pp. 888-889, Clive Tomlin, Ed., British Crop Protection Council, 49Downing Street, Farmham, Surrey G49 7PH, UNITED KINGDOM), andsulfonylcarboxamides (Alvarado et al., U.S. Pat. No. 4,883,914) act byinhibiting AHAS enzymatic activity. (See, Chaleff et al., Science224:1443, 1984; LaRossa et al., J.Biol.Chem. 259:8753, 1984; Ray, PlantPhysiol. 75:827, 11984; Shaner et al., Plant Physiol. 76:545, 1984).These herbicides are highly effective and environmentally benign. Theiruse in agriculture, however, is limited by their lack of selectivity,since crops as well as undesirable weeds are sensitive to the phytotoxiceffects of these herbicides.

Bedbrook et al., U.S. Pat. Nos. 5,013,659, 5,141,870, and 5,378,824,disclose several sulfonylurea resistant AHAS variants. However, thesevariants were either obtained by mutagenizing plants, seeds, or cellsand selecting for herbicide-resistant mutants, or were derived from suchmutants. This approach is unpredictable in that it relies (at leastinitially) on the random chance introduction of a relevant mutation,rather than a rational design approach based on a structural model ofthe target protein.

Thus, there is still a need in the art for methods and compositions thatprovide selective wide spectrum and/or specific herbicide resistance incultivated crops. The present inventors have discovered that selectiveherbicide resistant variant forms of AHAS and plants containing the samecan be prepared by structure-based modelling of AHAS against pyruvateoxidase (POX), identifying an herbicide binding pocket or pockets on theAHAS model, and designing specific mutations that alter the affinity ofthe herbicide for the binding pocket. These variants and plants are notinhibited or killed by one or more classes of herbicides and retainsufficient AHAS enzymatic activity to support crop growth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are illustrations of a 600 amino acid sequencecorresponding to the approximately 599 amino acid sequence ofacetohydroxy acid synthase (AHAS) from Zea mays (SEQ ID NO:1) which isgiven as an example of a plant AHAS enzyme. The sequence does notinclude a transit sequence, and the extra glycine is vestigial from athrombin cleavage site. Residues Met53, Arg128, and Phe135 are shown inbold.

FIGS. 2A and 2B are an illustration of the alignment of the sequence ofmaize AHAS and pyruvate oxidase (POX) from Lactobacillus planarum (SEQID NO:2).

FIGS. 3A-3C are schematic representations of the secondary structure ofan AHAS subunit. Regular secondary structure elements, α-helices andβ-sheets, are depicted as circles and ellipses, respectively, and arenumbered separately for each of the three domains within a subunit.Loops and coiled regions are represented by black lines, with numbersrepresenting the approximate beginnings and ends of the elements. Thelocations of cofactor binding sites and known mutation sites areindicated by octahedrons and stars, respectively.

FIG. 4 is an illustration of a computer-generated model of the activesite of maize AHAS with imazethapyr (PURSUIT® herbicide) modeled intothe binding pocket.

FIGS. 5A-5H are illustrations of the homology among AHAS amino acidsequences derived from different plant species. pAC 751 is maize als 2AHAS isozyme as expressed from the pAC 751 E. coil expression vector asin FIG. 1 (SEQ ID NO:1); Maize als 2 is the maize als 2 AHAS isozyme(SEQ ID NO:3); Maize. als 1 is the maize als 1 AHAS isozyme (SEQ IDNO:4); Tobac 1 is the tobacco AHAS SuRA isozyme (SEQ ID NO:5); Tobac 2is the tobacco AHAS SuRB isozyme (SEQ ID NO:6); Athcsr 12 is theArabidopsis thaliana Csr 1.2 AHAS gene (SEQ ID NO:7); Bnaal 3 is theBrassica napus AHAS III isozyme (SEQ ID NO:8); and Bnaal 2 is theBrassica napus AHAS II isozyme (SEQ ID NO:9).

FIG. 6 is a photographic illustration of an SDS-polyacrylamide gelstained for protein showing purification of maize AHAS. The lanescontain (from left to right): A, Molecular weight markers; B, Crude E.coli cell extract; C, Glutathione-agarose affinity purified preparation;D, Thrombin digest of the affinity purified preparation; E, Second passthrough glutathione-agarose column and Sephacryl S-100 gel filtration.

FIG. 7 is a graphic illustration of the results of in vitro assays ofthe enzymatic activity of wild-type and mutant AHAS proteins in theabsence and in the presence of increasing concentrations of imazethapyr(PURSUIT® herbicide). The Y axis represents the % of activity of themutant enzyme, wherein the 100% value is measured in the absence ofinhibitor.

FIG. 8 is a graphic illustration of the results of in vitro assays ofthe enzymatic activity of wild-type and mutant AHAS proteins in theabsence and presence of increasing concentrations of sulfometuron methyl(OUST® herbicide). The Y axis represents the % of activity of the mutantenzyme, wherein the 100% value is measured in the absence of inhibitor.

FIG. 9 is a graphic illustration of in vitro assays of the enzymaticactivity of wild-type Arabidopsis AHAS protein and the Met124Ile mutantArabidopsis AHAS protein in the absence and presence of increasingconcentrations of imazethapyr (PURSUIT® herbicide) and sulfometuronmethyl (OUST® herbicide). The Y axis represents the % activity of themutant enzyme, wherein the 100% value is measured in the absence ofinhibitor.

FIG. 10 is a graphic illustration of in vitro assays of the enzymaticactivity of wild-type Arabidopsis AHAS protein and the Met124His mutantArabidopsis AHAS protein in the absence and presence of increasingconcentrations of imazethapyr (PURSUIT® herbicide) and sulfometuronmethyl (OUST® herbicide). The Y axis represents the % activity of themutant enzyme, wherein the 100% value is measured in the absence ofinhibitor.

FIG. 11 is a graphic illustration of in vitro assays of the enzymaticactivity of wild-type Arabidopsis AHAS protein and Arg199Glu mutantArabidopsis AHAS protein in the absence and presence of increasingconcentrations of imazethapyr (PURSUIT® herbicide) and sulfometuronmethyl (OUST® herbicide). The Y axis represents the % activity of themutant enzyme, wherein the 100% value is measured in the absence ofinhibitor.

FIG. 12 is a schematic illustration of a DNA vector used for planttransformation, which contains the nptII gene (encoding kanamycinresistance) under the control of the 35S promoter and an AHAS gene (wildtype or variant) under the control of the Arabidopsis AHAS promoter.

FIG. 13 is a photograph showing the root development of tobacco plantstransformed with the Arabidopsis AHAS gene containing either theMet124Ile or Arg199Glu mutation and a non-transformed control. Plantswere grown for 18 days after transfer into medium containing 0.25 μMimazethapyr.

FIG. 14 is a photograph showing tobacco plants transformed with theArabidopsis AHAS gene containing either the Met124Ile, Met 124His, orArg199Glu mutation and a non-transformed control, which had been sprayedwith twice the field rate (100 g/ha) of imazethapyr.

FIG. 15 is a photograph showing the results of germination testsperformed in the presence of the herbicide CL 299,263 (imazamox), whichwere performed on seeds harvested from primary tobacco planttransformants that had been transformed with the Arabidopsis AHAS genecontaining either the Met124Ile, Met 124His, or Arg199Glu mutation.

SUMMARY OF THE INVENTION

The present invention provides a structure-based modelling method forthe production of herbicide resistant AHAS variant protein. The methodincludes:

(a) aligning a target AHAS protein on pyruvate oxidase template or anAHAS modelling equivalent thereof to derive the three-dimensionalstructure of the target AHAS protein;

(b) modelling one or more herbicides into the three-dimensionalstructure to localize an herbicide binding pocket in the target AHASprotein;

(c) selecting as a target for a mutation, at least one amino acidposition in the target AHAS protein, wherein the mutation alters theaffinity of at least one herbicide for the binding pocket;

(d) mutating DNA encoding the target AHAS protein to produce a mutatedDNA encoding a variant AHAS containing the mutation, such as, forexample, at least one different amino acid, at the position; and

(e) expressing the mutated DNA in a first cell, under conditions inwhich the variant AHAS containing the mutation, such as, for example,the different amino acid(s), at the position is produced.

The method further may include:

(f) expressing DNA encoding wild-type AHAS protein parallel in a secondcell;

(g) purifying the wild-type and the variant AHAS proteins from thecells;

(h) assaying the wild-type and the variant AHAS proteins for catalyticactivity in conversion of pyruvate to acetolactate or in thecondensation of pyruvate and 2-ketobutyrate to formacetohydroxybutyrate, in the absence and in the presence of theherbicide; and

(i) repeating steps (c)-(h), wherein the DNA encoding the AHAS variantof step (e) is used as the AHAS-encoding DNA in step (c) until a firstherbicide resistant AHAS variant protein is identified having:

(i) in the absence of the at least one herbicide,

(a) catalytic activity alone sufficient to maintain the viability of acell in which it is expressed; or

(b) catalytic activity in combination with any herbicide resistant AHASvariant protein also expressed in the cell, which may be the same as ordifferent than the first AHAS variant protein, sufficient to maintainthe viability of a cell in which it is expressed;

wherein the cell requires AHAS activity for viability; and

(ii) catalytic activity that is more resistant to the at least oneherbicide than is wild-type AHAS.

An alternate structure-based modelling method for the production ofherbicide resistant AHAS variant protein is also provided. This methodincludes:

(a) aligning a target AHAS protein on a first AHAS template derived froma polypeptide having the sequence of FIG. 1 or a functional equivalentthereof to derive the three-dimensional structure of the target AHASprotein;

(b) modelling one or more herbicides into the three-dimensionalstructure to localize an herbicide binding pocket in the target AHASprotein;

(c) selecting as a target for a mutation, at least one amino acidposition in the target AHAS protein, wherein the mutation alters theaffinity of at least one herbicide for the binding pocket;

(d) mutating DNA encoding the target AHAS protein to produce a mutatedDNA encoding a variant AHAS containing the mutation at the position; and

(e) expressing the mutated DNA in a first cell, under conditions inwhich the variant AHAS containing the mutation at the position isproduced.

This method can further include:

(f) expressing DNA encoding wild-type AHAS protein in parallel in asecond cell;

(g) purifying the wild-type and the variant AHAS protein from the cells;

(h) assaying the wild-type and the variant AHAS protein for catalyticactivity in conversion of pyruvate to acetolactate or in thecondensation of pyruvate and 2-ketobutyrate to formacetohydroxybutyrate, in the absence and in the presence of theherbicide; and

(i) repeating steps (c)-(h), wherein the DNA encoding the AHAS variantof step (e) is used as the AHAS-encoding DNA in step (c) until a firstherbicide resistant AHAS variant protein is identified having:

(i) in the absence of the at least one herbicide,

(a) catalytic activity alone sufficient to maintain the viability of acell in which it is expressed; or

(b) catalytic activity in combination with any herbicide resistant AHASvariant protein also expressed in the cell, which may be the same as ordifferent than the first AHAS variant protein, sufficient to maintainthe viability of a cell in which it is expressed;

wherein the cell requires AHAS activity for viability; and

(ii) catalytic activity that is more resistant to the at least oneherbicide than is wild-type AHAS.

In another alternate embodiment, the method includes:

(a) aligning a target AHAS protein on a first AHAS template having anidentified herbicide binding pocket and having the sequence of FIG. 1 ora functional equivalent thereof to derive the three-dimensionalstructure of the target AHAS protein;

(b) selecting as a target for a mutation, at least one amino acidposition in the target AHAS protein, wherein the mutation alters theaffinity of at least one herbicide for the binding pocket;

(c) mutating DNA encoding the target AHAS protein to produce a mutatedDNA encoding a variant AHAS containing the mutation at the position; and

(d) expressing the mutated DNA in a first cell, under conditions inwhich the variant AHAS containing the mutation at the position isproduced.

This method can further include:

(e) expressing DNA encoding wild-type target AHAS protein in parallel ina second cell;

(f) purifying the wild-type and the variant AHAS protein from the cells;

(g) assaying the wild-type and the variant AHAS protein for catalyticactivity in conversion of pyruvate to acetolactate or in thecondensation of pyruvate and 2-ketobutyrate to formacetohydroxybutyrate, in the absence and in the presence of theherbicide; and

(h) repeating steps (b)-(g), wherein the DNA encoding the AHAS variantof step (d) is used as the AHAS-encoding DNA in step (b) until a firstherbicide resistant AHAS variant protein is identified having:

(i) in the absence of the at least one herbicide,

(a) catalytic activity alone sufficient to maintain the viability of acell in which it is expressed; or

(b) catalytic activity in combination with any herbicide resistant AHASvariant protein also expressed in the cell, which may be the same as ordifferent than the first AHAS variant protein, sufficient to maintainthe viability of a cell in which it is expressed;

wherein the cell requires AHAS activity for viability; and

(ii) catalytic activity that is more resistant to the at least oneherbicide than is wild-type AHAS.

In preferred embodiments of the above methods, the catalytic activity inthe absence of the herbicide is at least about 5% and most preferably ismore than about 20% of the catalytic activity of the wild-type AHAS.Where the herbicide is an imidazolinone herbicide, the herbicideresistant AHAS variant protein preferably has:

(i) catalytic activity in the absence of the herbicide of more thanabout 20% of the catalytic activity of the wild-type AHAS;

(ii) catalytic activity that is relatively more resistant to thepresence of imidazolinone herbicides compared to wild-type AHAS; and

(iii) catalytic activity that is relatively more sensitive to thepresence of sulfonylurea herbicides compared to imidazolinoneherbicides.

The present invention further provides isolated DNA encodingacetohydroxy acid synthase (AHAS) variant proteins, the variant proteinscomprising an AHAS protein modified by:

(i) substitution of at least one different amino acid residue at anamino acid residue of the sequence of FIG. 1 (SEQ ID NO:1) selected fromthe group consisting of P48, G49, S52, M53, E54, A84, A95, T96, S97,G98, P99, G100, A101, V125, R127, R128, M129, I130, G131, T132, D133,F135, Q136, D186, I187, T259, T260, L261, M262, G263, R276, M277, L278,G279, H281, G282, T283, V284, G300, V301, R302, F303, D304, R306, V307,T308, G309, K310, I311, E312, A313, F314, A315, S316, R317, A318, K319,I320, E329, I330, K332, N333, K334, Q335, T404, G413, V414, G415, Q416,H417, Q418, M419, W420, A421, A422, L434, S435, S436, A437, G438, L439,G440, A441, M442, G443, D467, G468, S469, L471, N473, L477, M479, Q495,H496, L497, G498, M499, V501, Q502, Q504, D505, R506, Y508, K509, A510,N511, R512, A513, H514, T515, S524, H572, Q573, E574, H575, V576, L577,P578, M579, I580, P581, G583, G584, functional equivalents of any of theforegoing, and any combination of any of the foregoing;

(ii) deletion of up to 5 amino acid residues preceding, or up to 5 aminoacid residues following at least one amino acid residue of the sequenceof FIG. 1 (SEQ ID NO:1) selected from the group consisting of P48, G49,S52, M53, E54, A84, A95, T96, S97, G98, P99, G100, A101, V125, R127,R128, M129, I130, G131, T132, D133, F135, Q136, D186, I187, T259, T260,L261, M262, G263, R276, M277, L278, G279, H281, G282, T283, V284, G300,V301,R302,F303, D304, R306, V307, T308, G309, K310, I311, E312, A313,F314, A315, S316, R317, A318, K319, I320, E329, I330, K332, N333, K334,Q335, T404, G413, V414, G415, Q416, H417, Q418, M419, W420, A421, A422,L434, S435, S436, A437, G438, L439, G440, A441, M442, G443, D467, G468,S469, L471, N473, L477, M479, Q495, H496, L497, G498, M499, V501, Q502,Q504, D505, R506, Y508, K509, A510, N511, R512, A513, H514, T515, S524,H572, Q573, E574, H575, V576, L577, P578, M579, I580, P581, G583, G584,functional equivalents of any of the foregoing, and any combination ofany of the foregoing;

(iii) deletion of at least one amino acid residue or a functionalequivalent thereof between Q124 and H150 of the sequence of FIG. 1 (SEQID NO:1);

(iv) addition of at least one amino acid residue or a functionalequivalent thereof between Q124 and H150 of the sequence of FIG. 1 (SEQID NO:1);

(v) deletion of at least one amino acid residue or a functionalequivalent thereof between G300 and D324 of the sequence of FIG. 1 (SEQID NO:1);

(vi) addition of at least one amino acid residue or a functionalequivalent thereof between G300 and D324 of the sequence of FIG. 1 (SEQID NO:1); or

(vii) any combination of any of the foregoing.

In this numbering system, residue #2 corresponds to the putative aminoterminus of the mature protein, i.e., after removal of a chloroplasttargeting peptide.

The above modifications are directed to altering the ability of anherbicide, and preferably an imidazolinone-based herbicide, to inhibitthe enzymatic activity of the protein. In a preferred embodiment, theisolated DNA encodes an herbicide-resistant variant of AHAS. Alsoprovided are DNA vectors comprising DNA encoding these AHAS variants,variant AHAS proteins themselves, and cells, grown either in vivo or incell culture, that express the AHAS variants or comprise these vectors.

In another aspect, the present invention provides a method forconferring herbicide resistance on a cell or cells and particularly aplant cell or cells such as, for example, a seed. An AHAS gene,preferably the Arabidopsis thaliana AHAS gene, is mutated to alter theability of an herbicide to inhibit the enzymatic activity of the AHAS.The mutant gene is cloned into a compatible expression vector, and thegene is transformed into an herbicide-sensitive cell under conditions inwhich it is expressed at sufficient levels to confer herbicideresistance on the cell.

Also contemplated are methods for weed control, wherein a cropcontaining an herbicide resistant AHAS gene according to the presentinvention is cultivated and treated with a weed-controlling effectiveamount of the herbicide.

Also disclosed is a structure-based modelling method for the preparationof a first herbicide which inhibits AHAS activity. The method comprises:

(a) aligning a target AHAS protein on pyruvate oxidase template or anAHAS modelling functional equivalent thereof to derive thethree-dimensional structure of the target AHAS protein;

(b) modelling a second herbicide having AHAS inhibiting activity intothe three-dimensional structure to derive the location, structure, or acombination thereof of an herbicide binding pocket in the target AHASprotein; and

(c) designing a non-peptidic first herbicide which will interact with,and preferably will bind to, an AHAS activity inhibiting effectiveportion of the binding pocket, wherein the first herbicide inhibits theAHAS activity sufficiently to destroy the viability of a cell whichrequires AHAS activity for viability.

An alternative structure-based modelling method for the production of afirst herbicide which inhibits AHAS activity, is also enclosed. Themethod comprises:

(a) aligning a target AHAS protein on a first AHAS template derived froma polypeptide having the sequence of FIG. 1 or a functional equivalentthereof, to derive the three-dimensional structure of the target AHASprotein;

(b) modelling a second herbicide having AHAS inhibiting activity intothe three-dimensional structure to derive the location, structure, or acombination thereof of an herbicide binding pocket in the target AHASprotein; and

(c) designing a non-peptidic first herbicide which will interact with,and preferably will bind to, an AHAS activity inhibiting effectiveportion of the binding pocket, wherein the first herbicide inhibits theAHAS activity sufficiently to destroy the viability of a cell whichrequires AHAS activity for viability.

Preferably in each method, the first herbicide contains at least onefunctional group that interacts with a functional group of the bindingpocket.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses the rational design or structure-basedmolecular modelling of modified versions of the enzyme AHAS and AHASinhibiting herbicides. These modified enzymes (AHAS variant proteins)are resistant to the action of herbicides. The present invention alsoencompasses DNAs that encode these variants, vectors that include theseDNAs, the AHAS variant proteins, and cells that express these variants.Additionally provided are methods for producing herbicide resistance inplants by expressing these variants and methods of weed control. The DNAand the AHAS variants of the present invention were discovered instudies that were based on molecular modelling of the structure of AHAS.

Rational Structure-Based Design of AHAS Variants and AHAS InhibitingHerbicides

Herbicide-resistant variants of AHAS according to the present inventionare useful in conferring herbicide resistance in plants and can bedesigned with the POX model, AHAS model, or functional equivalentsthereof, such as, for example, transketolases, carboligases, pyruvatedecarboxylase, proteins that bind FAD and/or TPP as a cofactor, or anyproteins which have structural features similar to POX and/or AHAS; withan AHAS model such as a model having the sequence of FIG. 1 (SEQ IDNO:1); or with a functional equivalent of the sequence of FIG. 1 (SEQ IDNO:1) including a variant modeled from a previous model. Proteins thatcan be used include any proteins having less than a root mean squaredeviation of less than 3.5 angstroms in their Cα carbons relative to anyof the above-listed molecules. AHAS directed herbicides can be similarlymodelled from these templates. A functional equivalent of an AHAS aminoacid sequence is a sequence having substantial, i.e., 60-70%, homology,particularly in conserved regions such as, for example, a putativebinding pocket. The degree of homology can be determined by simplealignment based on programs known in the art, such as, for example, GAPand PILEUP by GCG. Homology means identical amino acids or conservativesubstitutions. A functional equivalent of a particular amino acidresidue in the AHAS protein of FIG. 1 (SEQ ID NO:1) is an amino acidresidue of another AHAS protein which when aligned with the sequence ofFIG. 1 (SEQ ID NO:1) by programs known in the art, such as, for example,GAP and PILEUP by GCG, is in the same position as the amino acid residueof FIG. 1 (SEQ ID NO:1).

Rational design steps typically include: (1) alignment of a target AHASprotein with a POX backbone or structure or an AHAS backbone orstructure; (2) optionally, and if the AHAS backbone has an identifiedherbicide binding pocket, modelling one or more herbicides into thethree-dimensional structure to localize an herbicide binding pocket inthe target protein; (3) selection of a mutation based upon the model;(4) site-directed mutagenesis; and (5) expression and purification ofthe variants. Additional steps can include (6) assaying of enzymaticproperties and (7) evaluation of suitable variants by comparison to theproperties of the wild-type AHAS. Each step is discussed separatelybelow.

1. Molecular Modelling

Molecular modelling (and particularly protein homology modelling)techniques can provide an understanding of the structure and activity ofa given protein. The structural model of a protein can be determineddirectly from experimental data such as x-ray crystallography,indirectly by homology modelling or the like, or combinations thereof(See White, et al., Annu. Rev. Biophys. Biomol. Struct., 23:349, 1994).Elucidation of the three-dimensional structure of AHAS provides a basisfor the development of a rational scheme for mutation of particularamino acid residues within AHAS that confer herbicide resistance on thepolypeptide.

Molecular modelling of the structure of Zea mays AHAS, using as atemplate the known X-ray crystal structure of related pyruvate oxidase(POX) from Lactobacillus plantarum, provides a three-dimensional modelof AHAS structure that is useful for the design of herbicide-resistantAHAS variants or AHAS inhibiting herbicides. This modelling proceduretakes advantage of the fact that AHAS and POX share a number ofbiochemical characteristics and may be derived from a common ancestralgene (Chang et al., J.Bacteriol. 170:3937, 1988).

Because of the high degree of cross-species homology in AHAS themodelled AHAS described herein or functional equivalents thereof canalso be used as templates for AHAS variant protein design.

Derivation of one model using interactive molecular graphics andalignments is described in detail below. The three-dimensional AHASstructure that results from this procedure predicts the approximatespatial organization of the active site of the enzyme and of the bindingsite or pocket of inhibitors such as herbicides including, but notlimited to, imidazolinone herbicides. The model is then refined andre-interpreted based on biochemical studies which are also describedbelow.

Protein homology modelling requires the alignment of the primarysequence of the protein under study with a second protein whose crystalstructure is known. Pyruvate oxidase (POX) was chosen for AHAS homologymodelling because POX and AHAS share a number of biochemicalcharacteristics. For example, both AHAS and POX share aspects ofenzymatic reaction mechanisms, as well as cofactor and metalrequirements. In both enzymes thiamine pyrophosphate (TPP), flavinadenine dinucleotide (FAD), and a divalent cation are required forenzymatic activity. FAD mediates a redox reaction during catalysis inPOX but presumably has only a structural function in AHAS, which ispossibly a vestigial remnant from the evolution of AHAS from POX. Bothenzymes utilize pyruvate as a substrate and form hydroxyethyl thiaminepyrophosphate as a stable reaction intermediate (Schloss, J. V. et al.In Biosynthesis of branched chain amino acids, Barak, Z. J. M., Chipman,D. M., Schloss, J. V. (eds) VCH Publishers, Weinheim, Germany, 1990).

Additionally, AHAS activity is present in chimeric POX-AHAS proteinsconsisting of the N-terminal half of POX and the C-terminal half ofAHAS, and there is a small degree of AHAS activity exhibited by POXitself. AHAS and POX also exhibit similar properties in solution (Risse,B. et al, Protein Sci. 1: 1699 and 1710, 1992; Singh, B. K., & Schmitt,G. K. (1989), FEBS Letters, 2: 113; Singh, B. K. et al. (1989) In:Prospects for Amino Acid Biosynthesis Inhibitors in Crop Protection andPharmaceutical Chemistry, (Lopping, L. G., et al., eds., BCPC Monographp. 87). With increasing protein concentration, both POX and AHAS undergostepwise transitions from monomers to dimers and tetramers. Increases inFAD concentration also induce higher orders of subunit assembly. Thetetrameric form of both proteins is most stable to heat and chemicaldenaturation.

Furthermore, the crystal structure of POX from Lactobacillus planarumhad been solved by Muller et al., Science 259:965, 1993. The presentinventors found that based in part upon the degree of physical,biochemical, and genetic homology between AHAS and POX, the X-raycrystal structure of POX could be used as a structural starting pointfor homology modelling of the AHAS structure.

AHAS and L. plantarum POX sequences were not similar enough for acompletely computerized alignment, however. Overall, only about 20% ofthe amino acids are identical, while about 50% of the residues are ofsimilar class (i.e. acidic, basic, aromatic, and the like). However, ifthe sequences are compared with respect to hydrophilic and hydrophobicresidue classifications, over 500 of the 600 amino acids match.Secondary structure predictions for AHAS (Holley et al.,Proc.Natl.Acad.Sci.USA 86:152, 1989) revealed a strong similarity to theactual secondary structure of POX. For nearly 70% of the residues, thepredicted AHAS secondary structure matches that of POX.

POX monomers consist of three domains, all having a central, parallelβ-sheet with crossovers consisting of α-helices and long loops. (Mulleret al., Science 259:965, 1993). The topology of the sheets differsbetween the domains, i.e. in the first and third domains, the strandsare assembled to the β-sheet in the sequence 2-1-3-4-6-5, while in theβ-sheet of the second domain, the sequence reads 3-2-1-4-5-6.

Computer generated alignments were based on secondary structureprediction and sequence homology. The conventional pair-wise sequencealignment method described by Needleman and Wunch, J. Mol. Biol, 48:443, 1970, was used. Two sequences were aligned to maximize thealignment score. The alignment score (homology score) is the sum of thescores for all pairs of aligned residues, plus an optional penalty forthe introduction of gaps into the alignment. The score for the alignmentof a pair of residues is a tabulated integer value. The homology scoringsystem is based on observing the frequency of divergence between a givenpair of residues. (MO Dayhoff, RM Schwartz & BC Orcutt “Atlas of ProteinSequence and Structure” vol. 5 suppl. 3 pp. 345-362, 1978).

The alignments were further refined by repositioning gaps so as toconserve continuous regular secondary structures. Amino acidsubstitutions generated by evaluation of likely alignment schemes werecompared by means of interactive molecular graphics. Alignments with themost conservative substitutions with respect to the particularfunctionality of the amino acids within a given site were chosen. Thefinal alignment of both POX and AHAS is displayed in FIG. 2. Conservedclusters of residues were identified, in particular for the TPP bindingsite and for parts of the FAD binding site. The alignment revealed ahigh similarity between AHAS and POX for the first domain, for mostparts of the second domain, and for about half of the third domain. Mostof the regions that aligned poorly and may fold differently in POX andin AHAS were expected to be at the surface of the protein and were notinvolved in cofactor or inhibitor binding. The prediction of mutationsites is not substantially affected by small shifts in the alignment.

Most TPP binding residues are highly conserved between POX and AHAS(e.g. P48-G49-G50). In some cases, residues that were close to TPPdiffer between POX and AHAS but remain within a region that is highlyconserved (for example, residues 90-110). On the other hand, the FADbinding site appeared to be less conserved. Although some FAD bindingresidues were strongly conserved (for example, D325-I326-D327-P328),others clearly differed between AHAS and POX (for example, residues inthe loop from positions 278 to 285 are not homologous. A detailedanalysis revealed that, at least for some of the less-conserved contactsites, the interactions were mediated by the polypeptide backbone ratherthan by the side chains. Hence, conservation was only required for thepolypeptide fold and was not required for the amino acid sequence (forexample, the backbone of residues 258-263 binds the ribitol chain ofFAD). One half of the adenine and the isoalloxazine binding sitesclearly differ.

After aligning the primary structure, a homology model was built bytransposition of AHAS amino acid sequences to the POX templatestructure. Missing coordinates were built stepwise using templates ofamino acid residues to complete undefined side chains. Data banksearches and energy minimization of small parts of the molecule wereused to complete the conformations of undefined loop regions. Thecofactors TPP and FAD were modeled into their binding pockets. Thismodel was then subjected to a complete, 5000 cycle energy minimization.All computer modelling was performed in an IRIS Indigo Elan R4000Workstation from Silicon Graphics Co. Interactive molecular modellingand energy-minimization were performed using Quanta/CHARMm 4.0 fromMolecular Simulations Inc. During this step, the conformation wasstable, indicating that no strongly disfavored interactions, such as,for example, close van der Waals contacts, had occurred. The results areshown schematically in FIG. 3.

Characteristics of Predicted AHAS Structure

Inspection of the modelled AHAS structure described above revealed thatmost of the protein folds with a backbone that is energeticallyreasonable, with most hydrophilic side chains accessible to the solvent.The surface of the β-sheets are smooth and accommodate the cross-overregions that are attached to them.

A model for dimeric AHAS was generated by duplicating the coordinates ofthe energy minimized monomeric AHAS and superimposing the two copies ontwo POX subunits using pairs of Cα coordinates as defined in thealignment scheme. The polypeptide chain of AHAS folds into threesimilarly folded domains composed of a six-stranded parallel β-sheetcore surrounded by long “loops” and α-helices. Two subunits areassembled such that the first domain of one subunit is in closeproximity to the cofactor-binding domains 2 and 3 of the other subunit.A solvent-filled space remains between the subunits at this site. Thispocket, which is defined by the confluence of the three domains, is theproposed entry site for the substrate. It is also proposed to be thebinding site for herbicides.

The inner surface of the binding pocket is outlined by the cofactors.The thiazol of TPP is positioned at the bottom of the pocket. Domain 3contributes to the inner surface of the pocket with a short α-helix thatpoints its axis towards the pyrophosphate of TPP, compensating thephosphate charges with its dipolar moment. This critical helix, whichstarts with G498, a “turn” residue in close contact with TPP, and whichends at F507, contains three known mutation sites for sulfonylurearesistance: V500, W503, and F507 (See, U.S. Pat. Nos. 5,013,659;5,141,870; and 5,378,824). In domain 1, the loop defined as P48-S52(between β-strand 2 and α-helix 2) faces W503, a mutation in whichconfers resistance to imidazolinones. Residues Y47 to G50 are also incontact with TPP. This loop is adjacent to P184-Q189, another turn,which connects the last strand of the β-sheet of domain 1 with aβ-strand that connects with domain 2. Within the pocket, near itsentrance, is a long region of domain 1 that interacts with acomplementary stretch of domain 2. Residues 125-129 and 133-137 ofdomain 1 and residues 304-313 of domain 2 are at the surface of thepocket. A turn consisting of T96-G100 is between loop 125-129 and TPP. Afurther stretch of domain 3 and two regions of domain 2 that line thebinding pocket are at the opposite corner of the pocket. Residues 572,575, 582, and 583 of domain 3 define the pocket surface on one side. Theremaining part of the interior of the pocket's surface is defined by FADand by a loop, L278-G282, that contacts the isoalloxazine ring of FAD.

The structural models of the AHAS protein can also be used for therational design of herbicides or AHAS inhibitors.

2. Modelling of Herbicides Into Binding Sites

Imazethapyr, the active imidazolinone in PURSUIT®, was positioned intoits proposed binding site using interactive molecular graphics (FIG. 4)and the software described above (FIG. 4). K185 was chosen as an“anchor” to interact with the charge of the carboxyl group. Theimidazolinone's NH—CO unit was placed to form hydrogen bonds to G50 andA51. This positioned the methyl substitute of imazethapyr close to V500on the backbone of the small α-helix. The isopropyl group is possiblybound by hydrophobic residues of the amino acids in the region ofresidues 125-135 that contribute to the inner surface of the pocket. Thepyridine ring is most probably “sandwiched” between A134 or F135, F507and W503. W503 also interacts with the imidazolinone ring system.

In a similar fashion, the sulfonylurea herbicides were modelled into asite that partially overlapped the described imidazolinone binding site.Overlap of sulfonylurea and imidazolinone binding sites was consistentwith competition binding experiments and with established mutant data,which show that the same mutation in maize, W503L, can confer resistanceto both herbicides. In these models, most of the known mutation sitesthat confer sulfonylurea herbicide resistance, i.e. G50, A51, K185,V500, W503, F507, are in close contact to the bound herbicides. P126 andA51 are required for keeping the K185 side chain in place by generatinga hydrophobic pore. S582, a site for specific imidazolinone resistance,is distant from the binding region and is located in the region wherethe homology is so poor that a change in the fold is expected. The FADbinding site apparently has low homology between AHAS and POX in thisregion; S582 is a residue that confers resistance in maize, and thatS582 and its adjacent residues are in close contact to the active sitepocket. It is proposed that FAD and the loop region encompassingresidues 278 to 285 move slightly away from the third domain, (downwardin FIG. 4) and that a loop that contains S582 folds into the spacebetween the helix at positions 499 to 507 and the loop at positions 278to 285. D305, another known resistance site, is close to FAD andmodulates the interaction between domains 1 and 2. M280 may either beinvolved in positioning of the helix at positions 498 to 507 or directlyin inhibitor binding. M280 and D305 could also be directly involved ininhibitor binding if domains 1 and 2 move slightly closer to each other.

3. Selection of Mutations

Specific amino acid residues are pinpointed as sites for theintroduction of mutations into the primary sequence of AHAS. These aminoacids are selected based upon their position in that if that amino acidresidue position is modified, there will be a resultant alteration (i.e.decline) in the affinity of an herbicide for the binding pocket. It isnot necessary that the mutation position reside in the binding pocket asamino acid residues outside the pocket itself can alter the pocketcharge or configuration. The selection of target sites for mutation isachieved using molecular models as described above. For exampleaccording to the model above, arginine at position 128 (designated R128in FIG. 1 using the single-letter code for amino acids) is located nearthe entrance to the substrate- and herbicide-binding pocket and has alarge degree of conformational freedom that may allow it to participatein transport of charged herbicides into the binding pocket. Therefore,this residue is substituted by alanine to remove both its charge and itslong hydrophobic side chain. (The resulting mutation is designatedR128A).

The mutations may comprise simple substitutions, which replace thewild-type sequence with any other amino acid. Alternatively, themutations may comprise deletions or additions of one or more aminoacids, preferably up to 5, at a given site. The added sequence maycomprise an amino acid sequence known to exist in another protein, ormay comprise a completely synthetic sequence. Furthermore, more than onemutation and/or more than one type of mutation may be introduced into asingle polypeptide.

4. Site-Directed Mutagenesis

The DNA encoding AHAS can be manipulated so as to introduce the desiredmutations. Mutagenesis is carried out using methods that are standard inthe art, as described in, for example, Higuchi, R., Recombinant PCR, InM. A. Innis, et al., eds, PCR Protocols: A Guide to Methods andApplications, Academic Press, pp. 177-183, 1990.

5. Expression and Purification of Variants

The mutated or variant AHAS sequence is cloned into a DNA expressionvector (see, e.g., Example 3) and is expressed in a suitable cell suchas, for example, E. coli. Preferably, the DNA encoding AHAS is linked toa transcription regulatory element, and the variant AHAS is expressed aspart of a fusion protein, for example, glutathione-S-transferase, tofacilitate purification (see Example 3 below). The variant AHAS is thenpurified using affinity chromatography or any other suitable methodknown in the art. “Purification” of an AHAS polypeptide refers to theisolation of the AHAS polypeptide in a form that allows its enzymaticactivity to be measured without interference by other components of thecell in which the polypeptide is expressed.

6. Assaying of Enzymatic Properties

The purified variant AHAS may be assayed for one or more of thefollowing three properties:

(a) specific or catalytic activity for conversion of pyruvate toacetolactate (expressed as units/mg pure AHAS, wherein a unit ofactivity is defined as 1 μmole acetolactate produced/hour), or forcondensation of pyruvate and 2-ketobutyrate to form acetohydroxybutyrate(expressed as units/mg pure AHAS, wherein a unit of activity is definedas 1μ mole acetohydroxybutyrate produced/hr.;

(b) level of inhibition by herbicide, such as, for example,imidazolinone (expressed as IC₅₀, the concentration at which 50% of theactivity of the enzyme is inhibited); and

(c) selectivity of resistance to the selected herbicide vs. otherherbicides. The selectivity index is defined as the fold resistance ofthe mutant to imidazolinones relative to the wild-type enzyme, dividedby the fold resistance of the same mutant to other herbicides alsorelative to the wild-type). Fold resistance to an herbicide relative tothe wild-type enzyme is expressed as the IC₅₀ of variant, divided by theIC₅₀ of the wild type. The selectivity index (S.I.) is thus representedby the following equation:${S.I.} = {\frac{{IC}_{50}{\text{~~of~~variant~~for~~herb.A}/{IC}_{50}}\text{~~of wild~~type~~for~~herb.A}}{{IC}_{50}{\text{~~of~~variant~~for~~herb.B}/{IC}_{50}}\text{~~of~~wild~~type~~for~~herb.B}}.}$

Suitable assay systems for making these determinations include, but arenot limited to, those described in detail in Example 4 below.

7.a. Evaluation of Suitable Variants

The enzymatic properties of variant AHAS polypeptides are compared tothe wild-type AHAS. Preferably, a given mutation results in an AHASvariant polypeptide that retains in vitro enzymatic activity towardspyruvate or pyruvate and 2-ketobutyrate, i.e., the conversion ofpyruvate to acetolactate or in the condensation of pyruvate and2-ketobutyrate to form acetohydroxybutyrate (and thus is expected to bebiologically active in vivo), while exhibiting catalytic activity thatis relatively more resistant to the selected herbicide(s) than iswild-type AHAS. Preferably, the variant AHAS exhibits:

(i) in the absence of the at least one herbicide,

(a) catalytic activity alone sufficient to maintain the viability of acell in which it is expressed; or

(b) catalytic activity in combination with any herbicide resistant AHASvariant protein also expressed in the cell, which may be the same as ordifferent than the first AHAS variant protein, sufficient to maintainthe viability of a cell in which it is expressed;

wherein the cell requires AHAS activity for viability; and

(ii) catalytic activity that is more resistant to the at least oneherbicide than is wild type AHAS;

and that is relatively more resistant to the herbicide(s) than iswild-type AHAS.

Therefore, any one specific AHAS variant protein need not have the totalcatalytic activity necessary to maintain the viability of the cell, butmust have some catalytic activity in an amount, alone or in combinationwith the catalytic activity of additional copies of the same AHASvariant and/or the catalytic activity of other AHAS variant protein(s),sufficient to maintain the viability of a cell that requires AHASactivity for viability. For example, catalytic activity may be increasedto minimum acceptable levels by introducing multiple copies of a variantencoding gene into the cell or by introducing the gene which furtherincludes a relatively strong promoter to enhance the production of thevariant.

More resistant means that the catalytic activity of the variant isdiminished by the herbicide(s), if at all, to a lesser degree thanwild-type AHAS catalytic activity is diminished by the herbicide(s).Preferred more resistant variant AHAS retains sufficient catalytic tomaintain the viability of a cell, plant, or organism wherein at the sameconcentration of the same herbicide(s), wild-type AHAS would not retainsufficient catalytic activity to maintain the viability of the cell,plant, or organism.

Preferably the catalytic activity in the absence of herbicide(s) is atleast about 5% and, most preferably, is more than about 20% of thecatalytic activity of the wild-type AHAS in the absence of herbicide(s).Most preferred AHAS variants are more resistant to imidazolinoneherbicides than to other herbicides, such as sulfonylurea-basedherbicides, though in some applications selectivity is neither needednor preferred.

In the case of imidazolinone-resistant variant AHAS, it is preferredthat the AHAS variant protein has

(i) catalytic activity in the absence of said herbicide of more thanabout 20% of the catalytic activity of said wild-type AHAS;

(ii) catalytic activity that is relatively more resistant to presence ofimidazolinone herbicides compared to wild type AHAS; and

(iii) catalytic activity that is relatively more sensitive to thepresence of sulfonylurea herbicides compared to imidazolinoneherbicides. Most preferred herbicide-resistant AHAS variants exhibit aminimum specific activity of about 20 units/mg, minimal or no inhibitionby imidazolinone, and a selectivity index ranging from about 1.3 toabout 3000 relative to other herbicides.

Without wishing to be bound by theory, it is believed that systematicand iterative application of this method to wild type or other targetAHAS protein will result in the production of AHAS variants having thedesired properties of high enzymatic activity as explained above andresistance to one or more classes of herbicides. For example, mutationof a wild-type AHAS sequence at a particular position to a given aminoacid may result in a mutant that exhibits a high degree of herbicideresistance but a significant loss of enzymatic activity towards pyruvateor pyruvate and 2-ketobutyrate. In a second application of the abovemethod, the starting or target AHAS polypeptide would then be thisvariant (in place of the wild-type AHAS). Rational design then involvessubstituting other amino acids at the originally mutated position and/oradding or deleting amino acids at selected points or ranges in theexpectation of retaining herbicide resistance but also maintaining ahigher level of enzymatic activity.

The structure-based rational design of herbicide resistant AHAS proteinsoffers many advantages over conventional approaches that rely on randommutagenesis and selection. For example, when substitution of aparticular amino acid with another requires substitution of more thanone nucleotide within the codon, the likelihood of this occurringrandomly is so low as to be impractical. By contrast, even double ortriple changes in nucleotide sequence within a codon can be easilyimplemented when suggested by a rational design approach. For example,one rationally designed mutation to confer selective imidazolinoneresistance requires a change from arginine to glutamate. Arginine isencoded by CGT, CGC, CGA, CGG, AGA, AGG, while glutamate is encoded byGAA and GAG. Since none of the arginine codons begins with GA, thismutation would require a double substitution of adjacent nucleotideswhich would occur so rarely using random mutagenesis as to beunpredictable and unrepeatable with any certainty of success. Althoughmutation frequency can be increased during random mutagenesis,alterations in nucleotide sequence would have an equal probability ofoccurring throughout the AHAS gene, in the absence of priorsite-direction of the mutations. This increases the chance of obtainingan irrelevant mutation that interferes with enzymatic activity.Similarly, it would be rare, using random mutagenesis, to find amultiple amino acid substitution, deletion, or substitution/deletionmutation that confers herbicide resistance while maintaining catalyticactivity. Deletion mutations that confer herbicide resistance would alsobe unlikely using a random mutagenesis approach. Deletions would need tobe limited to small regions and would have to occur in triplets so as toretain the AHAS reading frame in order to retain enzymatic activity.

However, with a rational structure-based approach, double amino acidsubstitution and/or deletion mutations are relatively easily achievedand precisely targeted. Furthermore, different mutagens used in randommutagenesis create specific types of mutations. For example, sodiumazide creates point substitution mutations in plants, while radiationtends to create deletions. Accordingly, two mutagenesis protocols wouldhave to be employed to obtain a multiple combinationsubstitution/deletion.

Finally, the present structure-based method for rational design ofherbicide-resistant AHAS variants allows for iterative improvement ofherbicide resistance mutations, a step that is not facilitated by randommutagenesis. Identification of a mutation site for herbicide resistanceby random mutagenesis may offer little, if any, predictive value forguiding further improvements in the characteristics of the mutant. Thepresent structure-based approach, on the other hand, allows improvementsto be implemented based on the position, environment, and function ofthe amino acid position in the structural model.

The iterative improvement method also allows the independentmanipulation of three important properties of AHAS: level of resistance,selectivity of resistance, and catalytic efficiency. For example,compensatory mutations can be designed in a predictive manner. If aparticular mutation has a deleterious effect on the activity of anenzyme, a second compensatory mutation may be used to restore activity.For example, a change in the net charge within a domain when a chargedresidue is introduced or lost due to a mutation can be compensated byintroducing a second mutation. Prediction of the position and type ofresidue(s) to introduce, delete, or substitute at the second site inorder to restore enzymatic activity requires a knowledge ofstructure-function relationships derived from a model such as thatdescribed herein.

7.b. Design of Non-Peptide Herbicides or AHAS Inhibitors

A chemical entity that alters and may fit into the active site of thetarget protein or bind in any position where it could inhibit activitymay be designed by methods known in the art, such as, for example,computer design programs that assist in the design of compounds thatspecifically interact with a receptor site.

An example of such a program is LUDI (Biosym Technologies—San Diego,Calif.) (see also, Lam, et al., Science 263:380, 1994; Thompson, et al.,J. Med. Chem., 37:3100, 1994).

The binding pocket and particularly the amino acid residues that havebeen identified as being involved as inhibitor binding can be used asanchor points for inhibitor design.

The design of site-specific herbicides is advantageous in the control ofweed species that may spontaneously develop herbicide resistance in thefield, particularly due to mutations in the AHAS gene.

Herbicide-Resistant AHAS Variants: DNA, Vectors, and Polypeptides

The present invention also encompasses isolated DNA molecules encodingvariant herbicide-resistant AHAS polypeptides. Genes encoding AHASpolypeptides according to the present invention may be derived from anyspecies and preferably a plant species, and mutations conferringherbicide resistance may be introduced at equivalent positions withinany of these AHAS genes. The equivalence of a given codon position indifferent AHAS genes is a function of both the conservation of primaryamino acid sequence and its protein and the retention of similarthree-dimensional structure. For example, FIG. 5 illustrates the highdegree of sequence homology between AHAS polypeptides derived fromdifferent plant species. These AHAS polypeptides exhibit at least about60 to about 70% overall homology. Without wishing to be bound by theory,it is believed that in regions of the polypeptide having a highlyconserved sequence, the polypeptide chain conformation will also bepreserved. Thus, it is possible to use an AHAS-encoding sequence fromone species for molecular modelling, to introduce mutations predictivelyinto an AHAS gene from a second species for initial testing anditerative improvement, and finally, to introduce the optimized mutationsinto AHAS derived from yet a third plant species for expression in atransgenic plant.

In one series of embodiment, these AHAS DNAs encode variants of an AHASpolypeptide and preferably of the maize AHAS polypeptide of FIG. 1 (SEQID NO:1) in which the polypeptide is modified by substitution at ordeletion preceding or following one or more of FIG. 1 (SEQ ID NO:1)amino acid residues P48, G49, S52, M53, E54, A84, A95, T96, S97, G98,P99, G100, A101, V125, R127, R128, M129, I130, G131, T132, D133, F135,Q136, D186, I187, T259, T260, L261, M262, G263, R276, M277, L278, G279,H281, G282, T283, V284, G300, V301, R302, F303, D304, R306, V307, T308,G309, K310, I311, E312, A313, F314, A315, S316, R317, A318, K319, I320,E329, I330, K332, N333, K334, Q335, T404, G413, V414, G415, Q416, H417,Q418, M419, W420, A421, A422, L434, S435, S436, A437, G438, L439, G440,A441, M442, G443, D467, G468, S469, L471, N473, L477, M479, Q495, H496,L497, G498, M499, V501, Q502, Q504, D505, R506, Y508, K509, A510, N511,R512, A513, H514, T515, S524, H572, Q573, E574, H575, V576, L577, P578,M579, I580, P581, G583, G584, functional equivalents of any of theforegoing; insertions or deletions between FIG. 1 (SEQ ID NO:1) Q124 andH150 or functional equivalents thereof; insertions or deletions betweenFIG. 1 (SEQ ID NO:1) G300 and D324 or functional equivalents thereof;and any combination of any of the foregoing thereof.

The mutations, whether introduced into the polypeptide of FIG. 1 (SEQ IDNO:1) or at equivalent positions in another plant AHAS gene, maycomprise alterations in DNA sequence that result in a simplesubstitution of any one or more other amino acids or deletions of up to5 amino acid residues proceeding or up to 5 amino acids residuesfollowing any of the residence listed above. Suitable amino acidsubstituents include, but are not limited to, naturally occurring aminoacids.

The mutations, whether introduced into the polypeptide of FIG. 1 or atequivalent positions in another plant AHAS gene, may comprisealterations in DNA sequence that result in a simple substitution of anyone or more other amino acids or deletions of up to 5 amino acidresidues proceeding or up to 5 amino acids residues following any of theresidence listed above. Suitable amino acid substituents include, butare not limited to, naturally occurring amino acids.

Alternatively, the mutations may comprise alterations in DNA sequencesuch that one or more amino acids are added or deleted in frame at theabove positions. Preferably, additions comprise about 3 to about 30nucleotides, and deletions comprise about 3 to about 30 nucleotides.Furthermore, a single mutant polypeptide may contain more than onesimilar or different mutation.

The present invention encompasses DNA and corresponding RNA sequences,as well as sense and antisense sequences. Nucleic acid sequencesencoding AHAS polypeptides may be flanked by natural AHAS regulatorysequences, or may be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenylation sequences, introns, 5′- and 3′-noncoding regions, andthe like. Furthermore, the nucleic acids can be modified to alterstability, solubility, binding affinity and specificity. For example,variant AHAS-encoding sequences can be selectively methylated. Thenucleic acid sequences of the present invention may also be modifiedwith a label capable of providing a detectable signal, either directlyor indirectly. Exemplary labels include radioisotopes, fluorescentmolecules, biotin, and the like.

The invention also provides vectors comprising nucleic acids encodingAHAS variants. A large number of vectors, including plasmid and fungalvectors, have been described for expression in a variety of eukaryoticand prokaryotic hosts. Advantageously, vectors may also include apromotor operably linked to the AHAS encoding portion. The encoded AHASmay be expressed by using any suitable vectors and host cells, usingmethods disclosed or cited herein or otherwise known to those skilled inthe relevant art. Examples of suitable vectors include withoutlimitation pBIN-based vectors, pBluescript vectors, and pGEM vectors.

The present invention also encompasses both variant herbicide-resistantAHAS polypeptides or peptide fragments thereof. As explained above, thevariant AHAS polypeptides may be derived from the maize polypeptideshown in FIG. 1 or from any plant or microbial AHAS polypeptide,preferably plant AHAS polypeptide. The polypeptides may be furthermodified by, for example, phosphorylation, sulfation, acylation,glycosylation, or other protein modifications. The polypeptides may beisolated from plants, or from heterologous organisms or cells(including, but not limited to, bacteria, yeast, insect, plant, andmammalian cells) into which the gene encoding a variant AHAS polypeptidehas been introduced and expressed. Furthermore, AHAS polypeptides may bemodified with a label capable of providing a detectable signal, eitherdirectly or indirectly, including radioisotopes, fluorescent compounds,and the like.

Chemical-resistant Plants and Plants Containing Variant AHAS Genes

The present invention encompasses transgenic cells, including, but notlimited to seeds, organisms, and plants into which genes encodingherbicide-resistant AHAS variants have been introduced. Non-limitingexamples of suitable recipient plants are listed in Table 1 below:

TABLE 1 RECIPIENT PLANTS COMMON NAME FAMILY LATIN NAME Maize GramineaeZea mays Maize, Dent Gramineae Zea mays dentiformis Maize, FlintGramineae Zea mays vulgaris Maize, Pop Gramineae Zea mays microspermaMaize, Soft Gramineae Zea mays amylacea Maize, Sweet Gramineae Zea maysamyleasaccharata Maize, Sweet Gramineae Zea mays saccharate Maize, WaxyGramineae Zea mays ceratina Wheat, Dinkel Pooideae Triticum speltaWheat, Durum Pooideae Triticum durum Wheat, English Pooideae Triticumturgidum Wheat, Large Pooideae Triticum spelta Spelt Wheat, PolishPooideae Triticum polonium Wheat, Poulard Pooideae Triticum turgidumWheat, Pooideae Triticum monococcum Singlegrained Wheat, Small PooideaeTriticum monococcum Spelt Wheat, Soft Pooideae Triticum aestivum RiceGramineae oryza sativa Rice, American Gramineae Zizania aquatica WildRice, Australian Gramineae Oryza australiensis Rice, Indian GramineaeZizania aquatica Rice, Red Gramineae Oryza glaberrima Rice, TuscaroraGramineae Zizania aquatica Rice, West Gramineae Oryza glaberrima AfricanBarley Pooideae Hordeum vulgare Barley, Pooideae Hordeum irregulareAbyssinian Intermediate, also Irregular Barley, Pooideae Hordeumspontaneum Ancestral Tworow Barley, Pooideae Hordeum trifurcatumBeardless Barley, Egyptian Pooideae Hordeum trifurcatum Barley, PooideaeHordeum vulgare fourrowed polystichon Barley, sixrowed Pooideae Hordeumvulgare hexastichon Barley, Tworowed Pooideae Hordeum distichon Cotton,Abroma Dicotyledoneae Abroma augusta Cotton, American MalvaceaeGossypium hirsutum Upland Cotton, Asiatic Malvaceae Gossypium arboreumTree, also Indian Tree Cotton, Malvaceae Gossypium barbadense Brazilian,also, brasiliense Kidney, and, Pernambuco Cotton, Levant MalvaceaeGossypium herbaceum Cotton, Long Malvaceae Gossypium barbadense Silk,also Long Sample, Sea Island Cotton, Mexican, Malvaceae Gossypiumhirsutum also Short Staple Soybean, Soya Leguminosae Glycine max Sugarbeet Chenopodiaceae Beta vulgaris altissima Sugar cane Woody-plantArenga pinnata Tomato Solanaceae Lycopersicon esculentum Tomato, CherrySolanaceae Lycopersicon esculentum cerasiforme Tomato, Common SolanaceaeLycopersicon esculentum commune Tomato, Currant Solanaceae Lycopersiconpimpinellifolium Tomato, Husk Solanaceae Physalis ixocarpa Tomato,Hyenas Solanaceae Solanum incanum Tomato, Pear Solanaceae Lycopersiconesculentum pyriforme Tomato, Tree Solanaceae Cyphomandra betacea PotatoSolanaceae Solanum tuberosum Potato, Spanish, Convolvulaceae Ipomoeabatatas Sweet potato Rye, Common Pooideae Secale cereale Rye, MountainPooideae Secale montanum Pepper, Bell Solanaceae Capsicum annuum grossumPepper, Bird, Solanaceae Capsicum annuum also Cayenne, minimum GuineaPepper, Bonnet Solanaceae Capsicum sinense Pepper, Solanaceae Capsicumannuum Bullnose, also grossum Sweet Pepper, Cherry Solanaceae Capsicumannuum cerasiforme Pepper, Cluster, Solanaceae Capsicum annuum also RedCluster fasciculatum Pepper, Cone Solanaceae Capsicum annuum conoidesPepper, Goat, Solanaceae Capsicum frutescens also Spur Pepper, LongSolanaceae Capsicum frutescens longum Pepper, Solanaceae Capsicum annuumOranamental Red, abbreviatum also Wrinkled Pepper, Tabasco SolanaceaeCapsicum annuum Red conoides Lettuce, Garden Compositae Lactuca salivaLettuce, Compositae Lactuca saliva Asparagus, also asparagina CeleryLettuce, Blue Compositae Lactuca perennis Lettuce, Blue, CompositaeLactuca pulchella also Chicory Lettuce, Compositae Lactuca salivaCabbage, also capitata Head Lettuce, Cos, Compositae Lactuca sativa alsoLongleaf, longifolia Romaine Lettuce, Compositae Lactuca sativa Crinkle,also crispa Curled, Cutting, Leaf Celery Umbelliferae Apium graveolensdulce Celery, Umbelliferae Apium graveolens Blanching, also dulce GardenCelery, Root, Umbelliferae Apium graveolens also rapaceum TurniprootedEggplant, Garden Solanaceae Solanum melongena Sorghum Sorghum All cropspecies Alfalfa Leguminosae Medicago sativum Carrot Umbelliferae Daucuscarota sativa Bean, Climbing Leguminosae Phaseolus vulgaris vulgarisBean, Sprouts Leguminosae Phaseolus aureus Bean, Brazilian LeguminosaeCanavalia ensiformis Broad Bean, Broad Leguminosae Vicia faba Bean,Common, Leguminosae Phaseolus vulgaris also French, White, Kidney Bean,Egyptian Leguminosae Dolichos lablab Bean, Long, also Leguminosae Vignasesquipedalis Yardlong Bean, Winged Leguminosae Psophocarpustetragonolobus Oat, also Avena Sativa Common, Side, Tree Oat, Black,also Avena Strigosa Bristle, Lopsided Oat, Bristle Avena Pea, alsoLeguminosae Pisum, sativum Garden, Green, sativum Shelling Pea,Blackeyed Leguminosae Vigna sinensis Pea, Edible Leguminosae Pisumsativum Podded axiphium Pea, Grey Leguminosae Pisum sativum speciosumPea, Winged Leguminosae Tetragonolobus purpureus Pea, WrinkledLeguminosae Pisum sativum medullare Sunflower Compositae Helianthusannuus Squash, Autumn, Dicotyledoneae Cucurbita maxima Winter Squash,Bush, Dicotyledoneae Cucurbita pepo also Summer melopepo Squash, TurbanDicotyledoneae Cucurbita maxima turbaniformis Cucumber DicotyledoneaeCucumis sativus Cucumber, Momordica charantia African, also BitterCucumber, Ecballium elaterium Squirting, also Wild Cucumber, WildCucumis anguria Poplar, Woody-Plant Populus trichocarpa CaliforniaPoplar, European Populus nigra Black Poplar, Gray Populus canescensPoplar, Lombardy Populus italica Poplar, Populus alba Silverleaf, alsoWhite Poplar, Western Populus trichocarpa Balsam Tobacco SolanaceaeNicotiana Arabidopsis Cruciferae Arabidopsis thaliana Thaliana TurfgrassLolium Turfgrass Agrostis Other families of turfgrass Clover Leguminosae

Expression of the variant AHAS polypeptides in transgenic plants confersa high level of resistance to herbicides including, but not limited to,imidazolinone herbicides such as, for example, imazethapyr (PURSUIT®),allowing the use of these herbicides during cultivation of thetransgenic plants.

Methods for the introduction of foreign genes into plants are known inthe art. Non-limiting examples of such methods include Agrobacteriuminfection, particle bombardment, polyethylene glycol (PEG) treatment ofprotoplasts, electroporation of protoplasts, microinjection,macroinjection, tiller injection, pollen tube pathway, dry seedimbibition, laser perforation, and electrophoresis. These methods aredescribed in, for example, B. Jenes et al., and S. W. Ritchie et al. InTransgenic Plants, Vol. 1, Engineering and Utilization, ed. S.-D. Kung,R. Wu, Academic Press, Inc., Harcourt Brace Jovanovich 1993; and L.Mannonen et al., Critical Reviews in Biotechnology, 14:287-310, 1994.

In a preferred embodiment, the DNA encoding a variant AHAS is clonedinto a DNA vector containing an antibiotic resistance marker gene, andthe recombinant AHAS DNA-containing plasmid is introduced intoAgrobacterium tumefaciens containing a Ti plasmid. This “binary vectorsystem” is described in, for example, U.S. Pat. No. 4,490,838, and in Anet al., Plant Mol.Biol.Manual A3:1-19 (1988). The transformedAgrobacterium is then co-cultivated with leaf disks from the recipientplant to allow infection and transformation of plant cells. Transformedplant cells are then cultivated in regeneration medium, which promotesthe formation of shoots, first in the presence of the appropriateantibiotic to select for transformed cells, then in the presence ofherbicide. In plant cells successfully transformed with DNA encodingherbicide-resistant AHAS, shoot formation occurs even in the presence oflevels of herbicide that inhibit shoot formation from non-transformedcells. After confirming the presence of variant AHAS DNA using, forexample, polymerase chain reaction (PCR) analysis, transformed plantsare tested for their ability to withstand herbicide spraying and fortheir capabilities for seed germination and root initiation andproliferation in the presence of herbicide.

Other Applications

The methods and compositions of the present invention can be used in thestructure-based rational design of herbicide-resistant AHAS variants,which can be incorporated into plants to confer selective herbicideresistance on the plants. Intermediate variants of AHAS (for example,variants that exhibit sub-optimal specific activity but high resistanceand selectivity, or the converse) are useful as templates for the designof second-generation AHAS variants that retain adequate specificactivity and high resistance and selectivity.

Herbicide resistant AHAS genes can be transformed into crop species insingle or multiple copies to confer herbicide resistance. Geneticengineering of crop species with reduced sensitivity to herbicides can:

(1) Increase the spectrum and flexibility of application of specificeffective and environmentally benign herbicides such as imidazolinoneherbicides;

(2) Enhance the commercial value of these herbicides;

(3) Reduce weed pressure in crop fields by effective use of herbicideson herbicide resistant crop species and a corresponding increase inharvest yields;

(4) Increase sales of seed for herbicide-resistant plants;

(5) Increase resistance to crop damage from carry-over of herbicidesapplied in a previous planting;

(6) Decrease susceptibility to changes in herbicide characteristics dueto adverse climate conditions; and

(7) Increase tolerance to unevenly or mis-applied herbicides.

For example, transgenic AHAS variant protein containing plants can becultivated. The crop can be treated with a weed controlling effectiveamount of the herbicide to which the AHAS variant transgenic plant isresistant, resulting in weed control in the crop without detrimentallyaffecting the cultivated crop.

The DNA vectors described above that encode herbicide-resistant AHASvariants can be further utilized so that expression of the AHAS variantprovides a selectable marker for transformation of cells by the vector.The intended recipient cells may be in culture or in situ, and the AHASvariant genes may be used alone or in combination with other selectablemarkers. The only requirement is that the recipient cell is sensitive tothe cytotoxic effects of the cognate herbicide. This embodiment takesadvantage of the relative low cost and lack of toxicity of, for example,imidazolinone-based herbicides, and may be applied in any system thatrequires DNA-mediated transformation.

Exemplification with respect to Preferred Embodiments

The following examples are intended to illustrate the present inventionwithout limitation.

EXAMPLE 1 Design of Herbicide-resistant AHAS Variants

Residues located close to the proposed herbicide binding site of themodel described in detail above were selected for mutagenesis in orderto design an active AHAS polypeptide with decreased herbicide bindingcapacity. Each site at the surface of the pocket was considered in termsof potential interactions with other residues in the pocket, as well aswith cofactors and herbicides. For example, addition of positivelycharged residue(s) is expected to interfere with the charge distributionwithin the binding site, resulting in a loss in affinity of binding of anegatively-charged herbicide.

Three residues were identified as most useful targets for mutagenesis:

(1) F135 was believed to interact with both the isoalloxazine ring ofFAD and with the aromatic group of the herbicides. In accordance withthe strategy of introducing more charged residues into the bindingpocket, this residue was changed to arginine.

(2) M53 contacts helix 498-507. This helix contains known herbicideresistance mutation sites and is also implicated in TPP binding.Furthermore, substitution of glutamic acid at position 53 was believedto favor an interaction with K185, reducing the affinity of K185 for thecarboxylate group of imazethapyr.

(3) R128 is located near the entrance to the pocket, where it wasbelieved to be involved in the initial transport of charged herbicidesinto the binding pocket. This residue was changed to alanine to removeboth its charge and its long hydrophobic side chain.

EXAMPLE 2 Site-directed Mutagenesis of AHAS to ProduceHerbicide-resistant Variants

The Arabidopsis AHAS gene was inserted in-frame to the 3′ end of thecoding region of the glutathione S-transferase gene in the pGEX-2Tvector (Pharmacia). Construction of the vector in this manner maintainedthe six amino acid thrombin recognition sequence at the junction of theexpressed glutathione-S-transferase (GST)/AHAS fusion protein. Thrombindigestion of the expressed fusion protein results in an AHAS proteinwith an N-terminal starting position at the end of the transit peptideat a putative transit peptide processing site, with a residualN-terminal glycine derived from the thrombin recognition site. The finalamino terminus of the cleaved AHAS protein consists ofGly-Ser-Ser-Ile-Ser. Site-directed mutations were introduced into theAHAS gene in this vector.

Site-directed mutations were constructed according to the PCR method ofHiguchi (Recombinant PCR. In MA Innis, et al. PCR Protocols: A Guide toMethods and Applications, Academic Press, San Diego, pp. 177-183, 1990).Two PCR products, each of which overlap the mutation site, wereamplified. The primers in the overlap region contained the mutation. Theoverlapping PCR amplified fragments were combined, denatured, andallowed to re-anneal together, producing two possible heteroduplexproducts with recessed 3′-ends. The recessed 3′-ends were extended byTaq DNA polymerase to produce a fragment that was the sum of the twooverlapping PCR products containing the desired mutation. A subsequentre-amplification of this fragment with only the two “outside” primersresulted in the enrichment of the full-length product. The productcontaining the mutation was then re-introduced into the Arabidopsis AHASgene in the pGEX-2T vector.

EXAMPLE 3 Expression and Purification of AHAS Variants

A. Methods

E. coli (DH5α) cells transformed with the pGEX-2T vector containingeither the maize wild type AHAS gene (vector designation pAC751), theArabidopsis Ser653Asn mutant, or the Arabidopsis Ile401Phe mutant weregrown overnight in LB broth containing 50 μg/mL ampicillin. Theovernight culture of E. coli was diluted 1:10 in 1 L LB, 50 μg/mLampicillin, and 0.1% v/v antifoam A. The culture was incubated at 37° C.with shaking until the OD₆₀₀ reached approximately 0.8.Isopropylthiogalactose (IPTG) was added to a final concentration of 1 mMand the culture was incubated for 3 more hours.

Cells were harvested by centrifugation at 8,670×g for 10 minutes in aJA-10 rotor and resuspended in {fraction (1/100)}th of the originalculture volume in MTPBS (16 mM Na₂HPO₄, 4 mM NaH₂PO₄, 150 mM NaCl, pH7.3). Triton X-100 and lysozyme were added to a final concentration of1% v/v and 100 μg/mL, respectively. Cells were incubated at 30° C. for15 minutes cooled to 4° C. on ice, and were lysed by sonication for 10seconds at level 7 with a Branson Sonifier Cell Disrupter equipped witha microtip probe. The cell free extract was centrifuged at 35,000×g for10 min. at 4° C. The supernatant was decanted and the centrifugationstep was repeated.

Purification of expressed fusion proteins was performed as modified fromSmith and Johnson (Gene 67:31-40, 1988). The supernatant was warmed toroom temperature and was passed through a 2 mL column ofglutathione-agarose beads (sulfur linkage, Sigma) equilibrated in MTPBS.The column was subsequently washed with MTPBS at room temperature untilthe A₂₈₀ of eluant matched that of MTPBS. The fusion protein was theneluted using a solution containing 5 mM reduced glutathione in 50 mMTris HCL, pH 8.0. The eluted fusion protein was treated withapproximately 30 NIH units of thrombin and dialyzed against 50 mMcitrate pH 6.5 and 150 mM NaCl.

The fusion protein was digested overnight at room temperature. Digestedsamples were dialyzed against MTPBS and passed twice through aglutathione-agarose column equilibrated in MTPBS to remove the releasedglutathione transferase protein. The protein fraction that did not bindto the column was collected and was concentrated by ultrafiltration on aYM10 filter (Amicon). The concentrated sample was loaded onto a 1.5×95cm Sephacryl S-100 gel filtration column equilibrated in gel filtrationbuffer (50 mM HEPES, 150 mM NaCl, pH 7.0). Two mL fractions werecollected at a flow rate of 0.14 mL/min. Enzyme stability was tested bystorage of the enzyme at 4° C. in gel filtration buffer with theaddition of 0.02% sodium azide and in the presence or absence of 2 mMthiamine pyrophosphate and 100 μM flavin adenine dinucleotide (FAD).

B. Results

E. coli transformed with the pAC751 plasmid containing the wide-typeAHAS gene fused downstream and in-frame with the GST gene expressed a 91kD protein when induced with IPTG. The 91 kD protein exhibited thepredicted molecular mass of a GST/AHAS fusion protein (the sum of 26 kDand 65 kD, respectively). When the cell free extract of DH5α/pAC751 waspassed through a glutathione-agarose affinity gel, washed, and elutedwith free glutathione it yielded a preparation enriched in the 91 kDprotein (FIG. 6, lane C). The six amino acid thrombin recognition siteengineered in the junction of GST and AHAS was successfully cleaved bythrombin (FIG. 6, lane D). The cleaved fusion protein preparationconsisted of the expected 26 kD GST protein and the 65 kD maize AHASprotein. Maize AHAS was purified to homogeneity by a second pass throughthe glutathione-agarose column to affinity subtract GST and subjected toa final Sephacryl S-100 gel filtration step to eliminated thrombin (FIG.6, lane E). The 65 kD protein is recognized on western blots by amonoclonal antibody raised against a maize AHAS peptide.

Purified wild type maize AHAS was analyzed by electrospray massspectrometry and was determined to have a molecular mass of 64,996daltons (data not shown). The predicted mass, as calculated from thededuced amino acid sequence of the gene inserted into the pGEX-2Tvector, is 65,058. The 0.096% discrepancy between the empiricallydetermined and predicted mass was within tuning variability of the massspectrometer. The close proximity of the two mass determinationssuggests that there were no misincorporated nucleotides duringconstruction of the expression vector, nor any post-translationalmodifications to the protein that would cause gross changes in molecularmass. Moreover, the lack of spurious peaks in the preparation ofpurified enzyme indicated that the sample was free of contamination.

EXAMPLE 4 Enzymatic Properties of AHAS Variants

The enzymatic properties of wild-type and variant AHAS produced in E.coli were measured by a modification of the method of Singh et al.(Anal. Biochem 211:173-179, 1988) as follows:

A reaction mixture containing 1× AHAS assay buffer (50 mM HEPES pH 7.0,100 mM pyruvate, 10 mM MgCl₂, 1 mM thiamine pyrophosphate (TPP), and 50μM flavin adenine dinucleotide (FAD)) was obtained either by dilution ofenzyme in 2× assay buffer or by addition of concentrated enzyme to 1×AHAS assay buffer. All assays containing imazethapyr and associatedcontrols contained a final concentration of 5% DMSO due to addition ofimazethapyr to assay mixtures as a 50% DMSO solution. Assays wereperformed in a final volume of 250 μL at 37° C. in microtiter plates.After allowing the reaction to proceed for 60 minutes, acetolactateaccumulation was measured calorimetrically as described by Singh et al.,Anal. Biochem 171:173-179, 1988.

Maize AHAS expressed and purified from pAC751 as described in Example 3above is active in the conversion of pyruvate to acetolactate. Full AHASactivity is dependent on the presence of the cofactors FAD and TPP inthe assay medium. No activity was detected when only FAD was added tothe assay medium. The activity of the purified enzyme with TPP only, orwith no cofactors, was less than 1% of the activity detected in thepresence of both TPP and FAD. Normally, AHAS present in crude plantextracts is very labile, particularly in the absence of substrate andcofactors. In contrast, the purified AHAS from the bacterial expressionsystem showed no lose in catalytic activity when stored for one month at4° C. in 50 mm HEPES pH 7.0, 150 mM NaCl, 0.02% NaN₃ in the presence orabsence of FAD and TPP. Furthermore, no degradation products werevisible from these stored preparations when resolved in SDS-PAGE gels.

The specific activities of wild-type AHAS and the M124E, R199A, andF206R variants are shown in Table 2 below. As determined from thealignment in FIG. 5, the M124E mutation in Arabidopsis AHAS is theequivalent of the maize M53E mutation, the R199A mutation in Arabidopsisis the equivalent of the maize R128A mutation, and the F206R mutation inArabidopsis is the equivalent of the maize F135R mutation. The mutationsdesigned in the maize AHAS structural model were used to identify theequivalent amino acid in the dicot Arabidopsis AHAS gene and wereincorporated and tested in the Arabidopsis AHAS gene. This translationand incorporation of rationally designed herbicide mutations into thedicot Arabidopsis AHAS gene can facilitate evaluation of herbicideresistance in plants of a dicot species.

TABLE 2 SPECIFIC ACTIVITY % Catalytic Activity as Compared to WildSpecific Activity Type Wild-Type 99.8 100 Met124Glu 9.15 9.16 Arg199Ala86.3 86.5 Phe206Arg 5.07 5.1

The R199A mutation maintains a high level of catalytic activity (Table2) while exhibiting a significant level of resistance to imazethapyr(FIG. 7). Notably, this variant retains complete sensitivity tosulfonylureas (FIG. 8). Thus, this variant fulfills the criteria of highspecific activity and selective herbicide resistance. By contrast, theM124E substitution resulted in almost complete resistance to imazethapyr(FIG. 7) but also exhibited severely reduced catalytic activity (Table2). Relative to imidazolinone resistance, this variant exhibits greatersensitivity to sulfonylurea (FIG. 8), suggesting that this residue is agood candidate for creating a mutation that confers selectiveresistance. Substitution of an amino acid other than glutamic acid mayhelp to maintain catalytic activity. The F206R substitution yieldedsimilar results to those observed with M124E variant, but lackedselectivity in resistance.

EXAMPLE 5 Iterative Improvement of AHAS Herbicide-Resistant VariantUsing a Rational Design Approach

Changing residue 124 in AHAS from Met to Glu as described in Example 4above conferred imidazolinone resistance but also reduced enzymaticactivity to 9.2% of the wild type value. The model of the maize AHASstructure described above suggested that Met53 (equivalent to theArabidopsis Met124 residue) interacts with a series of hydrophobicresidues on the face of an α-helix that is derived from a separatesubunit but are in close proximity to Met53. Thus, the hydrophobicinteraction between Met53 and the residues on the helix may stabilizeboth subunit/subunit association and the conformation of the activesite. It was believed that the substitution of the hydrophobic Metresidue with a charged glutamate residue most probably destabilizes theinter-subunit hydrophobic interaction and results in a loss of catalyticactivity.

Based on this structure/function analysis, the activity of the originalArabidopsis Met124Glu (equivalent to maize Met53Glu) mutant enzyme wasthen iteratively improved by substituting a more hydrophobic amino acid(Ile) at this position. The hydrophobic nature of the Ile side chainresulted in restoration of activity to wild type levels (specificactivity of 102, equivalent to 102% of the wild-type activity), but thegreater bulk of the Ile side chain was still able to maintain asignificant level of imidazolinone resistance (FIG. 9).

By comparison, substitution of a histidine residue at this positionresulting in an AHAS variant exhibiting a specific activity of 42.5,equivalent to 42.6% of the wild-type activity. This mutant, nonetheless,exhibited a high degree of resistance to PURSUIT® (FIG. 10).

EXAMPLE 6 Iterative Improvement of AHAS Herbicide-Resistant VariantUsing a Rational Design Approach

Another example of iterative refinement using the methods of the presentinvention involves the Arg128Ala variant. The structural model of maizeAHAS suggested that the Arg128 residue, which resides at the lip of theherbicide binding pocket, contributes to channeling charged substratesand herbicides into the herbicide binding pocket and into the activesite. The Arg 128 residue is distant from the TPP moiety, which bindsthe initial pyruvate molecule in the reaction mechanism of AHAS,explaining why the substitution of Arabidopsis AHAS Arg199 (theequivalent to maize Arg128) to alanine had little effect on thecatalytic activity of the enzyme. The structural model further indicatedthat a more radical change could be made at this position to raise thelevel of resistance while maintaining high levels of catalytic activity.On this basis, an iterative improvement of the mutation was made tosubstitute the positively charge arginine residue with a negativelycharged glutamate residue. The enzyme thus mutated had improved levelsof resistance to PURSUIT® while maintaining high levels of activity(specific activity of 114, equivalent to 114% of the wild-type activity)(FIG. 11).

EXAMPLE 7 Interchangeability of AHAS Derived from Different Species inStructure-Based Rational Design of Herbicide-Resistant AHAS Variants

A structural model of the three-dimensional structure of AHAS is builtwith a monocot AHAS sequence such as that derived from maize, asdescribed above. To introduce mutations into AHAS derived from a dicotspecies such as Arabidopsis, the sequences of AHAS derived from themonocot and dicot species are aligned using the GAP and PILEUP programs(Genetics Computer Group, 575 Sequence Drive, Madison, Wis. 53711).Equivalent positions are determined from the computer-generatedalignment. The mutations are then introduced into the dicot AHAS gene asdescribed above. Following expression of the mutant AHAS protein in E.coli and assessment of its biochemical properties (i.e., specificactivity and resistance to herbicides), the mutant gene is introducedinto a dicot plant by plant transformation methods as described above.

EXAMPLE 8 Production of Herbicide-Resistant Plants by Transformationwith Rationally Designed AHAS Genes

DNA Constructs:

Rationally designed AHAS variant genes contained within E. coliexpression vectors were used as a source of DNA restriction fragments toreplace the equivalent restriction fragment in a Arabidopsis AHAS gene.This gene is present in a 5.5 kb genomic DNA fragment which alsocontains the Arabidopsis AHAS promoter, the Arabidopsis AHAS terminationsequence and 5′- and 3′-flanking DNA. After DNA sequencing through themutation sites was performed to confirm the presence of the propermutation, the entire 5.5 kb fragment from each plasmid was inserted intoa pBIN based plant transformation vector (Mogen, Leiden, Netherlands).The plant transformation vector also contains the neomycinphosphotransferase II (nptII) kanamycin resistance gene driven by the35S cauliflower mosaic virus promoter. The final vector construct isdisplayed in FIG. 12. Vectors containing Arabidopsis AHAS genes withMet124Ile, Met124His, and Arg199Glu mutations (corresponding toMet53Ile, Met53His, and Arg128Glu mutations in the maize AHAS sequenceas shown in FIG. 1) were labeled pJK002, pJK003, and pJK004,respectively.

Each of these vectors was transformed into Agrobacterium tumefaciensstrain LBA4404 (R&D Life Technologies, Gaithersburg, Md.) using thetransformation method described in An et al., Plant Mol.Biol.ManualA3:1-19 (1988).

Plant Transformation:

Leaf disc transformation of Nicotiana tabacum cv. Wisconsin 38 wasperformed as described by Horsch et al. (Science, 227: 1229-1231, 1985)with slight modifications. Leaf discs were cut from plants grown understerile conditions and co-cultivated upsidedown in Murashige Skoog media(Sigma Chemical Co., St. Louis, Mo.) for 2-3 days at 25° C. in darknesswith Agrobacterium tumefaciens strains containing plasmids pJK002,pJK003, or pJK004. The discs were blotted dry and transferred toregeneration Murashige Skoog medium with B5 vitamins containing 1 mg/Lbenzyladenine and 0.1 mg/l 1-Napthyl Acetic Acid, 100 mg/L kanamycin,and 500 mg/L cefotaxime (all obtained from Sigma).

Initially, transformants were selected by kanamycin resistance conferredby the nptII gene present in the transformation vector. Shoots derivedfrom the leaf discs were excised and placed on fresh Murashige Skooghormone free media containing cefotaxime and kanamycin.

In Vivo Herbicide Resistance

Kanamycin-resistant tobacco shoots were transferred to medium containinga 0.25 μM imazethapyr. At this concentration of the imidazolinoneherbicide, non-transformed tobacco shoots (containing endogenouswild-type AHAS) were not able to initiate root formation. By contrast,root initiation and growth were observed from tobacco shoots transformedwith each of the mutant AHAS genes. Roots developed from shootstransformed with the Met124Ile and Arg199Glu mutant genes along withwild type are shown in FIG. 1. Furthermore, plants transformed with theMet124Ile or Arg199Glu mutant genes were resistant to spraying withtwice the field rate (100 g/ha) of imazethapyr (FIG. 13). The patternsof root growth in transformed vs. non-transformed plants in the presenceof herbicide, as well as the behavior after herbicide spraying suggestthat expression of the rationally designed herbicide resistance genesconfers herbicide resistance in vivo.

Detection of the Rationally Designed Genes in Herbicide ResistantTobacco

Genomic DNA was isolated from the AHAS-transformed tobacco plants, andthe presence of the Arabidopsis AHAS variant genes was verified by PCRanalysis. Differences between the nucleotide sequences of theArabidopsis AHAS gene and the two tobacco AHAS genes were exploited todesign PCR primers that amplify only the Arabidopsis gene in a tobaccogenomic DNA background. The rationally designed herbicide resistancegenes were detected, as shown by amplification of a DNA fragment of theproper size, in a majority of the herbicide resistant plants. No PCRsignal was seen from non-transformed tobacco plants.

Segregation of Transformed AHAS Genes:

To monitor segregation of rationally designed AHAS genes in transformedplants, germination tests were performed. Seeds were placed inhormone-free Murashige-Skoog medium containing up to 2.5 μM PURSUIT® and100 μM kanamycin. The seedlings that resulted were visually scored forresistance or susceptibility to the herbicide.

Since tobacco plants are diploid, it would be expected that the progenyof self-pollinated plants should segregate 3:1 resistant:susceptible,reflecting the existence of 1 seedling homozygous for the resistant AHASgene, 2 seedlings heterozygous for the resistant AHAS gene, and 1seedling lacking a resistant AHAS gene.

The results indicate that resistant AHAS genes are segregating in theexpected 3:1 ratio, supporting the conclusion that herbicide resistanceis conferred by a single, dominant copy of a rationally designed AHASgene.

These results indicate that rational design of herbicide-resistant AHASgenes can be used to produce plants that exhibit herbicide resistantgrowth in vivo.

EXAMPLE 9 Production of Plants Cross-Resistant to Different Herbicidesby Transformation with Rationally Designed AHAS Genes

Tobacco plants transformed with rationally designed AHAS genes asdescribed in Example 8 above were also tested for cross-resistance toanother herbicide, CL 299,263 (also known as imazamox). Germinationtests were performed on seeds harvested from the primary transformantscontaining the Met124Ile, Met124His, and Arg199Glu Arabidopsis AHASvariant genes, in the absence or presence of 2.5 μM CL 299,263 (FIG.15). This concentration of the herbicide causes severe stunting andbleaching of wild-type tobacco plants. Tobacco plants transformed withthe Met124His AHAS gene showed the greatest level of resistance (FIG.15). Arg199Glu transformants showed an intermediate level of resistance,while Met124Ile showed little resistance (FIG. 15).

All patents, applications, articles, publications, and test methodsmentioned above are hereby incorporated by reference.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed description.Such obvious variations are within the full intended scope of theappended claims.

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Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu 210 215 220 ValGly Glu Ser Arg Arg Pro Val Leu Tyr Val Gly Gly Gly Cys Ala 225 230 235240 Arg Ser Gly Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro 245250 255 Val Thr Thr Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro260 265 270 Leu Ser Leu Arg Met Leu Gly Met His Gly Thr Val Tyr Ala AsnTyr 275 280 285 Ala Val Asp Lys Ala Asp Leu Leu Leu Ala Leu Gly Val ArgPhe Asp 290 295 300 Asp Arg Val Thr Gly Lys Ile Glu Ala Phe Ala Ser ArgAla Lys Ile 305 310 315 320 Val His Val Asp Ile Asp Pro Ala Glu Ile GlyLys Asn Lys Gln Pro 325 330 335 His Val Ser Ile Cys Ala Asp Val Lys LeuAla Leu Gln Gly Met Asn 340 345 350 Ala Leu Leu Glu Gly Ser Thr Ser LysLys Ser Phe Asp Phe Gly Ser 355 360 365 Trp Asn Asp Glu Leu Asp Gln GlnLys Arg Glu Phe Pro Leu Gly Tyr 370 375 380 Lys Tyr Ser Asn Glu Glu IleGln Pro Gln Tyr Ala Ile Gln Val Leu 385 390 395 400 Asp Glu Leu Thr LysGly Glu Ala Ile Ile Gly Thr Gly Val Gly Gln 405 410 415 His Gln Met TrpAla Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln 420 425 430 Trp Leu SerSer Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala 435 440 445 Ala AlaGly Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile 450 455 460 AspGly Asp Gly Ser Phe Leu Met Asn Val Gln Glu Leu Ala Met Ile 465 470 475480 Arg Ile Glu Asn Leu Pro Val Lys Val Phe Val Leu Asn Asn Gln His 485490 495 Leu Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg500 505 510 Ala His Thr Tyr Leu Gly Asn Pro Glu Asn Glu Ser Glu Ile TyrPro 515 520 525 Asp Phe Val Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala ValArg Val 530 535 540 Thr Lys Lys Asn Glu Val Arg Ala Ala Ile Lys Lys MetLeu Glu Thr 545 550 555 560 Pro Gly Pro Tyr Leu Leu Asp Ile Ile Val ProHis Gln Glu His Val 565 570 575 Leu Pro Met Ile Pro Ser Gly Gly Ala PheLys Asp Met Ile Leu Asp 580 585 590 Gly Asp Gly Arg Thr Val Tyr 595 2585 PRT Lactobacillus planarum 2 Thr Asn Ile Leu Ala Gly Ala Ala Val IleLys Val Leu Glu Ala Trp 1 5 10 15 Gly Val Asp His Leu Tyr Gly Ile ProGly Gly Ser Ile Asn Ser Ile 20 25 30 Met Asp Ala Leu Ser Ala Glu Arg AspArg Ile His Tyr Ile Gln Val 35 40 45 Arg His Glu Glu Val Gly Ala Met AlaAla Ala Ala Asp Ala Lys Leu 50 55 60 Thr Gly Lys Ile Gly Val Cys Phe GlySer Ala Gly Pro Gly Gly Thr 65 70 75 80 His Leu Met Asn Gly Leu Tyr AspAla Arg Glu Asp His Val Pro Val 85 90 95 Leu Ala Leu Ile Gly Gln Phe GlyThr Thr Gly Met Asn Met Asp Thr 100 105 110 Phe Gln Glu Met Asn Glu AsnPro Ile Tyr Ala Asp Val Ala Asp Tyr 115 120 125 Asn Val Thr Ala Val AsnAla Ala Thr Leu Pro His Val Ile Asp Glu 130 135 140 Ala Ile Arg Arg AlaTyr Ala His Gln Gly Val Ala Val Val Gln Ile 145 150 155 160 Pro Val AspLeu Pro Trp Gln Gln Ile Ser Ala Glu Asp Trp Tyr Ala 165 170 175 Ser AlaAsn Asn Tyr Gln Thr Pro Leu Leu Pro Glu Pro Asp Val Gln 180 185 190 AlaVal Thr Arg Leu Thr Gln Thr Leu Leu Ala Ala Glu Arg Pro Leu 195 200 205Ile Tyr Tyr Gly Ile Gly Ala Arg Lys Ala Gly Lys Glu Leu Glu Gln 210 215220 Leu Ser Lys Thr Leu Lys Ile Pro Leu Met Ser Thr Tyr Pro Ala Lys 225230 235 240 Gly Ile Val Ala Asp Arg Tyr Pro Ala Tyr Leu Gly Ser Ala AsnArg 245 250 255 Val Ala Gln Lys Pro Ala Asn Glu Ala Leu Ala Gln Ala AspVal Val 260 265 270 Leu Phe Val Gly Asn Asn Tyr Pro Phe Ala Glu Val SerLys Ala Phe 275 280 285 Lys Asn Thr Arg Tyr Phe Leu Gln Ile Asp Ile AspPro Ala Lys Leu 290 295 300 Gly Lys Arg His Lys Thr Asp Ile Ala Val LeuAla Asp Ala Gln Lys 305 310 315 320 Thr Leu Ala Ala Ile Leu Ala Gln ValSer Glu Arg Glu Ser Thr Pro 325 330 335 Trp Trp Gln Ala Asn Leu Ala AsnVal Lys Asn Trp Arg Ala Tyr Leu 340 345 350 Ala Ser Leu Glu Asp Lys GlnGlu Gly Pro Leu Gln Ala Tyr Gln Val 355 360 365 Leu Arg Ala Val Asn LysIle Ala Glu Pro Asp Ala Ile Tyr Ser Ile 370 375 380 Asp Val Gly Asp IleAsn Leu Asn Ala Asn Arg His Leu Lys Leu Thr 385 390 395 400 Pro Ser AsnArg His Ile Thr Ser Asn Leu Phe Ala Thr Met Gly Val 405 410 415 Gly IlePro Gly Ala Ile Ala Ala Lys Leu Asn Tyr Pro Glu Arg Gln 420 425 430 ValPhe Asn Leu Ala Gly Asp Gly Gly Ala Ser Met Thr Met Gln Asp 435 440 445Leu Val Thr Gln Val Gln Tyr His Leu Pro Val Ile Asn Val Val Phe 450 455460 Thr Asn Cys Gln Tyr Gly Phe Ile Lys Asp Glu Gln Glu Asp Thr Asn 465470 475 480 Gln Asn Asp Phe Ile Gly Val Glu Phe Asn Asp Ile Asp Phe SerLys 485 490 495 Ile Ala Asp Gly Val His Met Gln Ala Phe Arg Val Asn LysIle Glu 500 505 510 Gln Leu Pro Asp Val Phe Glu Gln Ala Lys Ala Ile AlaGln His Glu 515 520 525 Pro Val Leu Ile Asp Ala Val Ile Thr Gly Asp ArgPro Leu Pro Ala 530 535 540 Glu Lys Leu Arg Leu Asp Ser Ala Met Ser SerAla Ala Asp Ile Glu 545 550 555 560 Ala Phe Lys Gln Arg Tyr Glu Ala GlnAsp Leu Gln Pro Leu Ser Thr 565 570 575 Tyr Leu Lys Gln Phe Gly Leu AspAsp 580 585 3 638 PRT Zea Mays 3 Met Ala Thr Ala Ala Ala Ala Ser Thr AlaLeu Thr Gly Ala Thr Thr 1 5 10 15 Ala Ala Pro Lys Ala Arg Arg Arg AlaHis Leu Leu Ala Thr Arg Arg 20 25 30 Ala Leu Ala Ala Pro Ile Arg Cys SerAla Ala Ser Pro Ala Met Pro 35 40 45 Met Ala Pro Pro Ala Thr Pro Leu ArgPro Trp Gly Pro Thr Asp Pro 50 55 60 Arg Lys Gly Ala Asp Ile Leu Val GluSer Leu Glu Arg Cys Gly Val 65 70 75 80 Arg Asp Val Phe Ala Tyr Pro GlyGly Ala Ser Met Glu Ile His Gln 85 90 95 Ala Leu Thr Arg Ser Pro Val IleAla Asn His Leu Phe Arg His Glu 100 105 110 Gln Gly Glu Ala Phe Ala AlaSer Gly Tyr Ala Arg Ser Ser Gly Arg 115 120 125 Val Gly Val Cys Ile AlaThr Ser Gly Pro Gly Ala Thr Asn Leu Val 130 135 140 Ser Ala Leu Ala AspAla Leu Leu Asp Ser Val Pro Met Val Ala Ile 145 150 155 160 Thr Gly GlnVal Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu 165 170 175 Thr ProIle Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu 180 185 190 ValLeu Asp Val Asp Asp Ile Pro Arg Val Val Gln Glu Ala Phe Phe 195 200 205Leu Ala Ser Ser Gly Arg Pro Gly Pro Val Leu Val Asp Ile Pro Lys 210 215220 Asp Ile Gln Gln Gln Met Ala Val Pro Val Trp Asp Lys Pro Met Ser 225230 235 240 Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys Pro Pro Ala Thr GluLeu 245 250 255 Leu Glu Gln Val Leu Arg Leu Val Gly Glu Ser Arg Arg ProVal Leu 260 265 270 Tyr Val Gly Gly Gly Cys Ala Ala Ser Gly Glu Glu LeuArg Arg Phe 275 280 285 Val Glu Leu Thr Gly Ile Pro Val Thr Thr Thr LeuMet Gly Leu Gly 290 295 300 Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu ArgMet Leu Gly Met His 305 310 315 320 Gly Thr Val Tyr Ala Asn Tyr Ala ValAsp Lys Ala Asp Leu Leu Leu 325 330 335 Ala Leu Gly Val Arg Phe Asp AspArg Val Thr Gly Lys Ile Glu Ala 340 345 350 Phe Ala Ser Arg Ala Lys IleVal His Val Asp Ile Asp Pro Ala Glu 355 360 365 Ile Gly Lys Asn Lys GlnPro His Val Ser Ile Cys Ala Asp Val Lys 370 375 380 Leu Ala Leu Gln GlyMet Asn Ala Leu Leu Glu Gly Ser Thr Ser Lys 385 390 395 400 Lys Ser PheAsp Phe Gly Ser Trp Asn Asp Glu Leu Asp Gln Gln Lys 405 410 415 Arg GluPhe Pro Leu Gly Tyr Lys Thr Ser Asn Glu Glu Ile Gln Pro 420 425 430 GlnTyr Ala Ile Gln Val Leu Asp Glu Leu Thr Lys Gly Glu Ala Ile 435 440 445Ile Gly Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Tyr Tyr 450 455460 Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser Ser Ala Gly Leu Gly Ala 465470 475 480 Met Gly Phe Gly Leu Pro Ala Ala Ala Gly Ala Ser Val Ala AsnPro 485 490 495 Gly Val Thr Val Val Asp Ile Asp Gly Asp Gly Ser Phe LeuMet Asn 500 505 510 Val Gln Glu Leu Ala Met Ile Arg Ile Glu Asn Leu ProVal Lys Val 515 520 525 Phe Val Leu Asn Asn Gln His Leu Gly Met Val ValGln Trp Glu Asp 530 535 540 Arg Phe Tyr Lys Ala Asn Arg Ala His Thr TyrLeu Gly Asn Pro Glu 545 550 555 560 Asn Glu Ser Glu Ile Tyr Pro Asp PheVal Thr Ile Ala Lys Gly Phe 565 570 575 Asn Ile Pro Ala Val Arg Val ThrLys Lys Asn Glu Val Arg Ala Ala 580 585 590 Ile Lys Lys Met Leu Glu ThrPro Gly Pro Tyr Leu Leu Asp Ile Ile 595 600 605 Val Pro His Gln Glu HisVal Leu Pro Met Ile Pro Ser Gly Gly Ala 610 615 620 Phe Lys Asp Met IleLeu Asp Gly Asp Gly Arg Thr Val Tyr 625 630 635 4 638 PRT Zea Mays 4 MetAla Thr Ala Ala Thr Ala Ala Ala Ala Leu Thr Gly Ala Thr Thr 1 5 10 15Ala Thr Pro Lys Ser Arg Arg Arg Ala His His Leu Ala Thr Arg Arg 20 25 30Ala Leu Ala Ala Pro Ile Arg Cys Ser Ala Leu Ser Arg Ala Thr Pro 35 40 45Thr Ala Pro Pro Ala Thr Pro Leu Arg Pro Trp Gly Pro Asn Glu Pro 50 55 60Arg Lys Gly Ser Asp Ile Leu Val Glu Ala Leu Glu Arg Cys Gly Val 65 70 7580 Arg Asp Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile His Gln 85 9095 Ala Leu Thr Arg Ser Pro Val Ile Ala Asn His Leu Phe Arg His Glu 100105 110 Gln Gly Glu Ala Phe Ala Ala Ser Ala Tyr Ala Arg Ser Ser Gly Arg115 120 125 Val Gly Val Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn LeuVal 130 135 140 Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser Val Pro Met ValAla Ile 145 150 155 160 Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr AspAla Phe Gln Glu 165 170 175 Thr Pro Ile Val Glu Val Thr Arg Ser Ile ThrLys His Asn Tyr Leu 180 185 190 Val Leu Asp Val Asp Asp Ile Pro Arg ValVal Gln Glu Ala Phe Phe 195 200 205 Leu Ala Ser Ser Gly Arg Pro Gly ProVal Leu Val Asp Ile Pro Lys 210 215 220 Asp Ile Gln Gln Gln Met Ala ValPro Ala Trp Asp Thr Pro Met Ser 225 230 235 240 Leu Pro Gly Tyr Ile AlaArg Leu Pro Lys Pro Pro Ala Thr Glu Phe 245 250 255 Leu Glu Gln Val LeuArg Leu Val Gly Glu Ser Arg Arg Pro Val Leu 260 265 270 Tyr Val Gly GlyGly Cys Ala Ala Ser Gly Glu Glu Leu Cys Arg Phe 275 280 285 Val Glu LeuThr Gly Ile Pro Val Thr Thr Thr Leu Met Gly Leu Gly 290 295 300 Asn PhePro Ser Asp Asp Pro Leu Ser Leu Arg Met Leu Gly Met His 305 310 315 320Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp Lys Ala Asp Leu Leu Leu 325 330335 Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys Ile Glu Ala 340345 350 Phe Ala Gly Arg Ala Lys Ile Val His Ile Asp Ile Asp Pro Ala Glu355 360 365 Ile Gly Lys Asn Lys Gln Pro His Val Ser Ile Cys Ala Asp ValLys 370 375 380 Leu Ala Leu Gln Gly Met Asn Thr Leu Leu Glu Gly Ser ThrSer Lys 385 390 395 400 Lys Ser Phe Asp Phe Gly Ser Trp His Asp Glu LeuAsp Gln Gln Lys 405 410 415 Arg Glu Phe Pro Leu Gly Tyr Lys Ile Phe AsnGlu Glu Ile Gln Pro 420 425 430 Gln Tyr Ala Ile Gln Val Leu Asp Glu LeuThr Lys Gly Glu Ala Ile 435 440 445 Ile Ala Thr Gly Val Gly Gln His GlnMet Trp Ala Ala Gln Tyr Tyr 450 455 460 Thr Tyr Lys Arg Pro Arg Gln TrpLeu Ser Ser Ala Gly Leu Gly Ala 465 470 475 480 Met Gly Phe Gly Leu ProAla Ala Ala Gly Ala Ala Val Ala Asn Pro 485 490 495 Gly Val Thr Val ValAsp Ile Asp Gly Asp Gly Ser Phe Leu Met Asn 500 505 510 Ile Gln Glu LeuAla Met Ile Arg Ile Glu Asn Leu Pro Val Lys Val 515 520 525 Phe Val LeuAsn Asn Gln His Leu Gly Met Val Val Gln Trp Glu Asp 530 535 540 Arg PheTyr Lys Ala Asn Arg Ala His Thr Phe Leu Gly Asn Pro Glu 545 550 555 560Asn Glu Ser Glu Ile Tyr Pro Asp Phe Val Ala Ile Ala Lys Gly Phe 565 570575 Asn Ile Pro Ala Val Arg Val Thr Lys Lys Ser Glu Val His Ala Ala 580585 590 Ile Lys Lys Met Leu Glu Ala Pro Gly Pro Tyr Leu Leu Asp Ile Ile595 600 605 Val Pro His Gln Glu His Val Leu Pro Met Ile Pro Ser Gly GlyAla 610 615 620 Phe Lys Asp Met Ile Leu Asp Gly Asp Gly Arg Thr Val Tyr625 630 635 5 667 PRT Tobacco 5 Met Ala Ala Ala Ala Pro Ser Pro Ser SerSer Ala Phe Ser Lys Thr 1 5 10 15 Leu Ser Pro Ser Ser Ser Thr Ser SerThr Leu Leu Pro Arg Ser Thr 20 25 30 Phe Pro Phe Pro His His Pro His LysThr Thr Pro Pro Pro Leu His 35 40 45 Leu Thr His Thr His Ile His Ile HisSer Gln Arg Arg Arg Phe Thr 50 55 60 Ile Ser Asn Val Ile Ser Thr Asn GlnLys Val Ser Gln Thr Glu Lys 65 70 75 80 Thr Glu Thr Phe Val Ser Arg PheAla Pro Asp Glu Pro Arg Lys Gly 85 90 95 Ser Asp Val Leu Val Glu Ala LeuGlu Arg Glu Gly Val Thr Asp Val 100 105 110 Phe Ala Tyr Pro Gly Gly AlaSer Met Glu Ile His Gln Ala Leu Thr 115 120 125 Arg Ser Ser Ile Ile ArgAsn Val Leu Pro Arg His Glu Gln Gly Gly 130 135 140 Val Phe Ala Ala GluGly Tyr Ala Arg Ala Thr Gly Phe Pro Gly Val 145 150 155 160 Cys Ile AlaThr Ser Gly Pro Gly Ala Thr Asn Leu Val Ser Gly Leu 165 170 175 Ala AspAla Leu Leu Asp Ser Val Pro Ile Val Ala Ile Thr Gly Gln 180 185 190 ValPro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile 195 200 205Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu Val Met Asp 210 215220 Val Glu Asp Ile Pro Arg Val Val Arg Glu Ala Phe Phe Leu Ala Arg 225230 235 240 Ser Gly Arg Pro Gly Pro Ile Leu Ile Asp Val Pro Lys Asp IleGln 245 250 255 Gln Gln Leu Val Ile Pro Asp Trp Asp Gln Pro Met Arg LeuPro Gly 260 265 270 Tyr Met Ser Arg Leu Pro Lys Leu Pro Asn Glu Met LeuLeu Glu Gln 275 280 285 Ile Val Arg Leu Ile Ser Glu Ser Lys Lys Pro ValLeu Tyr Val Gly 290 295 300 Gly Gly Cys Ser Gln Ser Ser Glu Asp Leu ArgArg Phe Val Glu Leu 305 310 315 320 Thr Gly Ile Pro Val Ala Ser Thr LeuMet Gly Leu Gly Ala Phe Pro 325 330 335 Thr Gly Asp Glu Leu Ser Leu SerMet Leu Gly Met His Gly Thr Val 340 345 350 Tyr Ala Asn Tyr Ala Val AspSer Ser Asp Leu Leu Leu Ala Phe Gly 355 360 365 Val Arg Phe Asp Asp ArgVal Thr Gly Lys Leu Glu Ala Phe Ala Ser 370 375 380 Arg Ala Lys Ile ValHis Ile Asp Ile Asp Ser Ala Glu Ile Gly Lys 385 390 395 400 Asn Lys GlnPro His Val Ser Ile Cys Ala Asp Ile Lys Leu Ala Leu 405 410 415 Gln GlyLeu Asn Ser Ile Leu Glu Ser Lys Glu Gly Lys Leu Lys Leu 420 425 430 AspPhe Ser Ala Trp Arg Gln Glu Leu Thr Glu Gln Lys Val Lys His 435 440 445Pro Leu Asn Phe Lys Thr Phe Gly Asp Ala Ile Pro Pro Gln Tyr Ala 450 455460 Ile Gln Val Leu Asp Glu Leu Thr Asn Gly Asn Ala Ile Ile Ser Thr 465470 475 480 Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Tyr Tyr Lys TyrArg 485 490 495 Lys Pro Arg Gln Trp Leu Thr Ser Gly Gly Leu Gly Ala MetGly Phe 500 505 510 Gly Leu Pro Ala Ala Ile Gly Ala Ala Val Gly Arg ProAsp Glu Val 515 520 525 Val Val Asp Ile Asp Gly Asp Gly Ser Phe Ile MetAsn Val Gln Glu 530 535 540 Leu Ala Thr Ile Lys Val Glu Asn Leu Pro ValLys Ile Met Leu Leu 545 550 555 560 Asn Asn Gln His Leu Gly Met Val ValGln Trp Glu Asp Arg Phe Tyr 565 570 575 Lys Ala Asn Arg Ala His Thr TyrLeu Gly Asn Pro Ser Asn Glu Ala 580 585 590 Glu Ile Phe Pro Asn Met LeuLys Phe Ala Glu Ala Cys Gly Val Pro 595 600 605 Ala Ala Arg Val Thr HisArg Asp Asp Leu Arg Ala Ala Ile Gln Lys 610 615 620 Met Leu Asp Thr ProGly Pro Tyr Leu Leu Asp Val Ile Val Pro His 625 630 635 640 Gln Glu HisVal Leu Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp 645 650 655 Val IleThr Glu Gly Asp Gly Arg Ser Ser Tyr 660 665 6 664 PRT Tobacco 6 Met AlaAla Ala Ala Ala Ala Pro Ser Pro Ser Phe Ser Lys Thr Leu 1 5 10 15 SerSer Ser Ser Ser Lys Ser Ser Thr Leu Leu Pro Arg Ser Thr Phe 20 25 30 ProPhe Pro His His Pro His Lys Thr Thr Pro Pro Pro Leu His Leu 35 40 45 ThrPro Thr His Ile His Ser Gln Arg Arg Arg Phe Thr Ile Ser Asn 50 55 60 ValIle Ser Thr Thr Gln Lys Val Ser Glu Thr Gln Lys Ala Glu Thr 65 70 75 80Phe Val Ser Arg Phe Ala Pro Asp Glu Pro Arg Lys Gly Ser Asp Val 85 90 95Leu Val Glu Ala Leu Glu Arg Glu Gly Val Thr Asp Val Phe Ala Tyr 100 105110 Pro Gly Gly Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Ser 115120 125 Ile Ile Arg Asn Val Leu Pro Arg His Glu Gln Gly Gly Val Phe Ala130 135 140 Ala Glu Gly Tyr Ala Arg Ala Thr Gly Phe Pro Gly Val Cys IleAla 145 150 155 160 Thr Ser Gly Pro Gly Ala Thr Asn Leu Val Ser Gly LeuAla Asp Ala 165 170 175 Leu Leu Asp Ser Val Pro Ile Val Ala Ile Thr GlyGln Val Pro Arg 180 185 190 Arg Met Ile Gly Thr Asp Ala Phe Gln Glu ThrPro Ile Val Glu Val 195 200 205 Thr Arg Ser Ile Thr Lys His Asn Tyr LeuVal Met Asp Val Glu Asp 210 215 220 Ile Pro Arg Val Val Arg Glu Ala PhePhe Leu Ala Arg Ser Gly Arg 225 230 235 240 Pro Gly Pro Val Leu Ile AspVal Pro Lys Asp Ile Gln Gln Gln Leu 245 250 255 Val Ile Pro Asp Trp AspGln Pro Met Arg Leu Pro Gly Tyr Met Ser 260 265 270 Arg Leu Pro Lys LeuPro Asn Glu Met Leu Leu Glu Gln Ile Val Arg 275 280 285 Leu Ile Ser GluSer Lys Lys Pro Val Leu Tyr Val Gly Gly Gly Cys 290 295 300 Ser Gln SerSer Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile 305 310 315 320 ProVal Ala Ser Thr Leu Met Gly Leu Gly Ala Phe Pro Thr Gly Asp 325 330 335Glu Leu Ser Leu Ser Met Leu Gly Met His Gly Thr Val Tyr Ala Asn 340 345350 Tyr Ala Val Asp Ser Ser Asp Leu Leu Leu Ala Phe Gly Val Arg Phe 355360 365 Asp Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala Lys370 375 380 Ile Val His Ile Asp Ile Asp Ser Ala Glu Ile Gly Lys Asn LysGln 385 390 395 400 Pro His Val Ser Ile Cys Ala Asp Ile Lys Leu Ala LeuGln Gly Leu 405 410 415 Asn Ser Ile Leu Glu Ser Lys Glu Gly Lys Leu LysLeu Asp Phe Ser 420 425 430 Ala Trp Arg Gln Glu Leu Thr Val Gln Lys ValLys Tyr Pro Leu Asn 435 440 445 Phe Lys Thr Phe Gly Asp Ala Ile Pro ProGln Tyr Ala Ile Gln Val 450 455 460 Leu Asp Glu Leu Thr Asn Gly Ser AlaIle Ile Ser Thr Gly Val Gly 465 470 475 480 Gln His Gln Met Trp Ala AlaGln Tyr Tyr Lys Tyr Arg Lys Pro Arg 485 490 495 Gln Trp Leu Thr Ser GlyGly Leu Gly Ala Met Gly Phe Gly Leu Pro 500 505 510 Ala Ala Ile Gly AlaAla Val Gly Arg Pro Asp Glu Val Val Val Asp 515 520 525 Ile Asp Gly AspGly Ser Phe Ile Met Asn Val Gln Glu Leu Ala Thr 530 535 540 Ile Lys ValGlu Asn Leu Pro Val Lys Ile Met Leu Leu Asn Asn Gln 545 550 555 560 HisLeu Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn 565 570 575Arg Ala His Thr Tyr Leu Gly Asn Pro Ser Asn Glu Ala Glu Ile Phe 580 585590 Pro Asn Met Leu Lys Phe Ala Glu Ala Cys Gly Val Pro Ala Ala Arg 595600 605 Val Thr His Arg Asp Asp Leu Arg Ala Ala Ile Gln Lys Met Leu Asp610 615 620 Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile Val Pro His Gln GluHis 625 630 635 640 Val Leu Pro Met Ile Pro Ser Gly Gly Ala Phe Lys AspVal Ile Thr 645 650 655 Glu Gly Asp Gly Arg Ser Ser Tyr 660 7 671 PRTArabidopsis thaliana 7 Met Ala Ala Ala Thr Thr Thr Thr Thr Thr Ser SerSer Ile Ser Phe 1 5 10 15 Ser Thr Lys Pro Ser Pro Ser Ser Ser Lys SerPro Leu Pro Ile Ser 20 25 30 Arg Phe Ser Leu Pro Phe Ser Leu Asn Pro AsnLys Ser Ser Ser Ser 35 40 45 Ser Arg Arg Arg Gly Ile Lys Ser Ser Ser ProSer Ser Ile Ser Ala 50 55 60 Val Leu Asn Thr Thr Thr Asn Val Thr Thr ThrPro Ser Pro Thr Lys 65 70 75 80 Pro Thr Lys Pro Glu Thr Phe Ile Ser ArgPhe Ala Pro Asp Gln Pro 85 90 95 Arg Lys Gly Ala Asp Ile Leu Val Glu AlaLeu Glu Arg Gln Gly Val 100 105 110 Glu Thr Val Phe Ala Tyr Pro Gly GlyAla Ser Met Glu Ile His Gln 115 120 125 Ala Leu Thr Arg Ser Ser Ser IleArg Asn Val Leu Pro Arg His Glu 130 135 140 Gln Gly Gly Val Phe Ala AlaGlu Gly Tyr Ala Arg Ser Ser Gly Lys 145 150 155 160 Pro Gly Ile Cys IleAla Thr Ser Gly Pro Gly Ala Thr Asn Leu Val 165 170 175 Ser Gly Leu AlaAsp Ala Leu Leu Asp Ser Val Pro Leu Val Ala Ile 180 185 190 Thr Gly GlnVal Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu 195 200 205 Thr ProIle Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu 210 215 220 ValMet Asp Val Glu Asp Ile Pro Arg Ile Ile Glu Glu Ala Phe Phe 225 230 235240 Leu Ala Thr Ser Gly Arg Pro Gly Pro Val Leu Val Asp Val Pro Lys 245250 255 Asp Ile Gln Gln Gln Leu Ala Ile Pro Asn Trp Glu Gln Ala Met Arg260 265 270 Leu Pro Gly Tyr Met Ser Arg Met Pro Lys Pro Pro Glu Asp SerHis 275 280 285 Leu Glu Gln Ile Val Arg Leu Ile Ser Glu Ser Lys Lys ProVal Leu 290 295 300 Tyr Val Gly Gly Gly Cys Leu Asn Ser Ser Asp Glu LeuGly Arg Phe 305 310 315 320 Val Glu Leu Thr Gly Ile Pro Val Ala Ser ThrLeu Met Gly Leu Gly 325 330 335 Ser Tyr Pro Cys Asp Asp Glu Leu Ser LeuHis Met Leu Gly Met His 340 345 350 Gly Thr Val Tyr Ala Asn Tyr Ala ValGlu His Ser Asp Leu Leu Leu 355 360 365 Ala Phe Gly Val Arg Phe Asp AspArg Val Thr Gly Lys Leu Glu Ala 370 375 380 Phe Ala Ser Arg Ala Lys IleVal His Ile Asp Ile Asp Ser Ala Glu 385 390 395 400 Ile Gly Lys Asn LysThr Pro His Val Ser Val Cys Gly Asp Val Lys 405 410 415 Leu Ala Leu GlnGly Met Asn Lys Val Leu Glu Asn Arg Ala Glu Glu 420 425 430 Leu Lys LeuAsp Phe Gly Val Trp Arg Asn Glu Leu Asn Val Gln Lys 435 440 445 Gln LysPhe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro Pro 450 455 460 GlnTyr Ala Ile Lys Val Leu Asp Glu Leu Thr Asp Gly Lys Ala Ile 465 470 475480 Ile Ser Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Phe Tyr 485490 495 Asn Tyr Lys Lys Pro Arg Arg Gln Trp Leu Ser Ser Gly Gly Leu Gly500 505 510 Ala Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala Ser Val AlaAsn 515 520 525 Pro Asp Ala Ile Val Val Asp Ile Asp Gly Asp Gly Ser PheIle Met 530 535 540 Asn Val Gln Glu Leu Ala Thr Ile Arg Val Glu Asn LeuPro Val Lys 545 550 555 560 Val Leu Leu Leu Asn Asn Gln His Leu Gly MetVal Met Gln Trp Glu 565 570 575 Asp Arg Phe Tyr Lys Ala Asn Arg Ala HisThr Phe Leu Gly Asp Pro 580 585 590 Ala Gln Glu Asp Glu Ile Phe Pro AsnMet Leu Leu Phe Ala Ala Ala 595 600 605 Cys Gly Ile Pro Ala Ala Arg ValThr Lys Lys Ala Asp Leu Arg Glu 610 615 620 Ala Ile Gln Thr Met Leu AspThr Pro Gly Pro Tyr Leu Leu Asp Val 625 630 635 640 Ile Cys Pro His GlnGlu His Val Leu Pro Met Ile Pro Asn Gly Gly 645 650 655 Thr Phe Asn AspVal Ile Thr Glu Gly Asp Gly Arg Ile Lys Tyr 660 665 670 8 652 PRTBrassica napus 8 Met Ala Ala Ala Thr Ser Ser Ser Pro Ile Ser Leu Thr AlaLys Pro 1 5 10 15 Ser Ser Lys Ser Pro Leu Pro Ile Ser Arg Phe Ser LeuPro Phe Ser 20 25 30 Leu Thr Pro Gln Lys Pro Ser Ser Arg Leu His Arg ProLeu Ala Ile 35 40 45 Ser Ala Val Leu Asn Ser Pro Val Asn Val Ala Pro GluLys Thr Asp 50 55 60 Lys Ile Lys Thr Phe Ile Ser Arg Tyr Ala Pro Asp GluPro Arg Lys 65 70 75 80 Gly Ala Asp Ile Leu Val Glu Ala Leu Glu Arg GlnGly Val Glu Thr 85 90 95 Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu IleHis Gln Ala Leu 100 105 110 Thr Arg Ser Ser Thr Ile Arg Asn Val Leu ProArg His Glu Gln Gly 115 120 125 Gly Val Phe Ala Ala Glu Gly Tyr Ala ArgSer Ser Gly Lys Pro Gly 130 135 140 Ile Cys Ile Ala Thr Ser Gly Pro GlyAla Thr Asn Leu Val Ser Gly 145 150 155 160 Leu Ala Asp Ala Met Leu AspSer Val Pro Leu Val Ala Ile Thr Gly 165 170 175 Gln Val Pro Arg Arg MetIle Gly Thr Asp Ala Phe Gln Glu Thr Pro 180 185 190 Ile Val Glu Val ThrArg Ser Ile Thr Lys His Asn Tyr Leu Val Met 195 200 205 Asp Val Asp AspIle Pro Arg Ile Val Gln Glu Ala Phe Phe Leu Ala 210 215 220 Thr Ser GlyArg Pro Gly Pro Val Leu Val Asp Val Pro Lys Asp Ile 225 230 235 240 GlnGln Gln Leu Ala Ile Pro Asn Trp Asp Gln Pro Met Arg Leu Pro 245 250 255Gly Tyr Met Ser Arg Leu Pro Gln Pro Pro Glu Val Ser Gln Leu Gly 260 265270 Gln Ile Val Arg Leu Ile Ser Glu Ser Lys Arg Pro Val Leu Tyr Val 275280 285 Gly Gly Gly Ser Leu Asn Ser Ser Glu Glu Leu Gly Arg Phe Val Glu290 295 300 Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly SerTyr 305 310 315 320 Pro Cys Asn Asp Glu Leu Ser Leu Gln Met Leu Gly MetHis Gly Thr 325 330 335 Val Tyr Ala Asn Tyr Ala Val Glu His Ser Asp LeuLeu Leu Ala Phe 340 345 350 Gly Val Arg Phe Asp Asp Arg Val Thr Gly LysLeu Glu Ala Phe Ala 355 360 365 Ser Arg Ala Lys Ile Val His Ile Asp IleAsp Ser Ala Glu Ile Gly 370 375 380 Lys Asn Lys Thr Pro His Val Ser ValCys Gly Asp Val Lys Leu Ala 385 390 395 400 Leu Gln Gly Met Asn Lys ValLeu Glu Asn Arg Ala Glu Glu Leu Lys 405 410 415 Leu Asp Phe Gly Val TrpArg Ser Glu Leu Ser Glu Gln Lys Gln Lys 420 425 430 Phe Pro Leu Ser PheLys Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr 435 440 445 Ala Ile Gln ValLeu Asp Glu Leu Thr Gln Gly Lys Ala Ile Ile Ser 450 455 460 Thr Gly ValGly Gln His Gln Met Trp Ala Ala Gln Phe Tyr Lys Tyr 465 470 475 480 ArgLys Pro Arg Gln Trp Leu Ser Ser Ser Gly Leu Gly Ala Met Gly 485 490 495Phe Gly Leu Pro Ala Ala Ile Gly Ala Ser Val Ala Asn Pro Asp Ala 500 505510 Ile Val Val Asp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn Val Gln 515520 525 Glu Leu Ala Thr Ile Arg Val Glu Asn Leu Pro Val Lys Ile Leu Leu530 535 540 Leu Asn Asn Gln His Leu Gly Met Val Met Gln Trp Glu Asp ArgPhe 545 550 555 560 Tyr Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asp ProAla Arg Glu 565 570 575 Asn Glu Ile Phe Pro Asn Met Leu Gln Phe Ala GlyAla Cys Gly Ile 580 585 590 Pro Ala Ala Arg Val Thr Lys Lys Glu Glu LeuArg Glu Ala Ile Gln 595 600 605 Thr Met Leu Asp Thr Pro Gly Pro Tyr LeuLeu Asp Val Ile Cys Pro 610 615 620 His Gln Glu His Val Leu Pro Met IlePro Ser Gly Gly Thr Phe Lys 625 630 635 640 Asp Val Ile Thr Glu Gly AspGly Arg Thr Lys Tyr 645 650 9 637 PRT Brassica napus 9 Met Ala Ser PheSer Phe Phe Gly Thr Ile Pro Ser Ser Pro Thr Lys 1 5 10 15 Ala Ser ValPhe Ser Leu Pro Val Ser Val Thr Thr Leu Pro Ser Phe 20 25 30 Pro Arg ArgArg Ala Thr Arg Val Ser Val Ser Ala Asn Ser Lys Lys 35 40 45 Asp Gln AspArg Thr Ala Ser Arg Arg Glu Asn Pro Ser Thr Phe Ser 50 55 60 Ser Lys TyrAla Pro Asn Val Pro Arg Ser Gly Ala Asp Ile Leu Val 65 70 75 80 Glu AlaLeu Glu Arg Gln Gly Val Asp Val Val Phe Ala Tyr Pro Gly 85 90 95 Gly AlaSer Met Glu Ile His Gln Ala Leu Thr Arg Ser Asn Thr Ile 100 105 110 ArgAsn Val Leu Pro Arg His Glu Gln Gly Gly Ile Phe Ala Ala Glu 115 120 125Gly Tyr Ala Arg Ser Ser Gly Lys Pro Gly Ile Cys Ile Ala Thr Ser 130 135140 Gly Pro Gly Ala Met Asn Leu Val Ser Gly Leu Ala Asp Ala Leu Phe 145150 155 160 Asp Ser Val Pro Leu Ile Ala Ile Thr Gly Gln Val Pro Arg ArgMet 165 170 175 Ile Gly Thr Met Ala Phe Gln Glu Thr Pro Val Val Glu ValThr Arg 180 185 190 Thr Ile Thr Lys His Asn Tyr Leu Val Met Glu Val AspAsp Ile Pro 195 200 205 Arg Ile Val Arg Glu Ala Phe Phe Leu Ala Thr SerVal Arg Pro Gly 210 215 220 Pro Val Leu Ile Asp Val Pro Lys Asp Val GlnGln Gln Phe Ala Ile 225 230 235 240 Pro Asn Trp Glu Gln Pro Met Arg LeuPro Leu Tyr Met Ser Thr Met 245 250 255 Pro Lys Pro Pro Lys Val Ser HisLeu Glu Gln Ile Leu Arg Leu Val 260 265 270 Ser Glu Ser Lys Arg Pro ValLeu Tyr Val Gly Gly Gly Cys Leu Asn 275 280 285 Ser Ser Glu Glu Leu ArgArg Phe Val Glu Leu Thr Gly Ile Pro Val 290 295 300 Ala Ser Thr Phe MetGly Leu Gly Ser Tyr Pro Cys Asp Asp Glu Glu 305 310 315 320 Phe Ser LeuGln Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr 325 330 335 Ala ValGlu Tyr Ser Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp 340 345 350 AspArg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala Lys Ile 355 360 365Val His Ile Asp Ile Asp Ser Thr Glu Ile Gly Lys Asn Lys Thr Pro 370 375380 His Val Ser Val Cys Cys Asp Val Gln Leu Ala Leu Gln Gly Met Asn 385390 395 400 Glu Val Leu Glu Asn Arg Arg Asp Val Leu Asp Phe Gly Glu TrpArg 405 410 415 Cys Glu Leu Asn Glu Gln Arg Leu Lys Phe Pro Leu Arg TyrLys Thr 420 425 430 Phe Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln LeuLeu Asp Glu 435 440 445 Leu Thr Asp Gly Lys Ala Ile Ile Thr Thr Gly ValGly Gln His Gln 450 455 460 Met Trp Ala Ala Gln Phe Tyr Arg Phe Lys LysPro Arg Gln Trp Leu 465 470 475 480 Ser Ser Gly Gly Leu Gly Ala Met GlyPhe Gly Leu Pro Ala Ala Met 485 490 495 Gly Ala Ala Ile Ala Asn Pro GlyAla Val Val Val Asp Ile Asp Gly 500 505 510 Asp Gly Ser Phe Ile Met AsnIle Gln Glu Leu Ala Thr Ile Arg Val 515 520 525 Glu Asn Leu Pro Val LysVal Leu Leu Ile Asn Asn Gln His Leu Gly 530 535 540 Met Val Leu Gln TrpGlu Asp His Phe Tyr Ala Ala Asn Arg Ala Asp 545 550 555 560 Ser Phe LeuGly Asp Pro Ala Asn Pro Glu Ala Val Phe Pro Asp Met 565 570 575 Leu LeuPhe Ala Ala Ser Cys Gly Ile Pro Ala Ala Arg Val Thr Arg 580 585 590 ArgGlu Asp Leu Arg Glu Ala Ile Gln Thr Met Leu Asp Thr Pro Gly 595 600 605Pro Phe Leu Leu Asp Val Val Cys Pro His Gln Asp His Val Leu Pro 610 615620 Leu Ile Pro Ser Gly Gly Thr Phe Lys Asp Ile Ile Val 625 630 635

What is claimed is:
 1. A variant plant acetohydroxy acid synthase (AHAS) protein comprising at least one mutation at an amino acid residue corresponding to an amino acid residue selected from the group consisting of M53, R128, I330, and any combination of the foregoing, of SEQ ID NO:1, where said variant plant AHAS protein is more resistant to an herbicide than a wild-type AHAS protein.
 2. A variant AHAS protein as defined in claim 1, wherein said herbicide is selected from the group consisting of an imidazolinones, sulfonylureas, triazolopyrimidine, sulfomamides, pyrimidyl-oxy-benzoic acids, sulfamoylureas, sulfonylcarboxamides, and combinations thereof.
 3. A variant AHAS protein as defined in claim 1, wherein said AHAS protein is derived from Arabidopsis thaliana.
 4. A variant AHAS protein as defined in claim 1, wherein said substitution is selected from the group consisting of Met53Trp, Met53Glu, Met53Ile, Met53His, Arg128Ala, Arg128Glu, Ile330Phe, an identical substitution at an amino acid residue of another plant AHAS protein at an amino acid position aligned with any of the foregoing, or a combination of any of the foregoing.
 5. A variant AHAS protein as defined in claim 1, wherein said variant AHAS protein has catalytic activity that is more resistant to at least one herbicide than is wild type AHAS.
 6. A variant AHAS protein as defined in claim 1, wherein said variant AHAS has more than about 20% of the catalytic activity of wild-type AHAS.
 7. A variant AHAS protein as defined in claim 1, wherein said variant AHAS is at least 2-fold more resistant to imidazolinone-based herbicides than to sulfonylurea-based herbicides. 